Enhancement of tumor response to chemotherapy by activation of the asmase/ceramide pathway through timed administration of a short-acting anti-angiogenic agent

ABSTRACT

Disclosed is a method for enhancing tumor response to chemotherapy, the method comprising administering a short-acting anti-angiogenic agent (AAA) capable of activating ASMase to a subject afflicted with a solid tumor, and thereby creating a time interval of increased susceptibility of said tumor to one or more chemotherapeutic agents, followed by administration of at least one chemotherapeutic agent within the interval. The interval can be defined in terms of a short-duration activation of ASMase signaling by the AAA. Disclosed are also methods for predicting the tumor response in a patient afflicted with a solid tumor to a chemotherapeutic agent, using as an indicator of the response ASMase level or activity (or ceramide level) in the patient following the administration of the chemotherapeutic agent to the patient, or dynamic IVIM based DW-MRI to measure perfusion alterations following administration of the chemotherapeutic agent.

STATEMENT OR RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/525,856, filed May 10, 2017, which is a U.S. National Stageof PCT Application No. PCT/US2015/060486, filed Nov. 12, 2015, whichclaims the benefit of and priority to U.S. Provisional PatentApplication No. 62/078,280, filed Nov. 11, 2014, the entire contents ofeach of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under CA105125 andCA158367 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE DISCLOSURE Technical Field

The present disclosure relates to the field of cancer therapy usinganti-angiogenic agents, not as anticancer therapeutics per se, but aschemosensitization agents—to make patients more sensitive tochemotherapy, thereby increasing the effect of chemotherapeutic drugs.The present disclosure also relates to the development of biomarkers tomonitor dosage, timing and effectiveness of such combined therapy.

Background and Description of Related Art

The main approaches to cancer treatment include radiation therapy,surgery, chemotherapy, immunotherapy and hormonal therapy.Chemotherapeutic agents can be grouped into several general classesbased on their mechanism of action: taxanes, alkylating agents,antitumor antibiotics, topoisomerase inhibitors (e.g., topoisomerase IIinhibitors), endoplasmic reticulum stress inducing agents,antimetabolites, and mitotic inhibitors.

While chemotherapeutic agents can be of substantial therapeutic benefitin many patients, their effectiveness is limited in many types ofcancer. Moreover, chemotherapy resistance remains a major hindrance incancer treatment. In order to improve clinical outcomes, a deeperunderstanding of the mechanisms that regulate chemotherapy sensitivityand resistance is necessary. Furthermore, the development of biomarkersthat could be used to predict the efficacy of chemotherapy and tooptimize dosage and administration regimens could contributesignificantly to such improved outcomes.

Angiogenesis, a process whereby new blood vessels are formed from thepre-existing ones, is a hallmark of tumor development and metastasis.During tumorigenesis, as cancer cells rapidly proliferate, tumors expandbeyond the support capacity of the existing vasculature, leading tohypoxia, depletion of nutrients and accumulation of metabolic wastes.Tumor cells in turn adapt to these conditions by upregulatingpro-angiogenic factors, such as vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), and platelet-derivedendothelial growth factor (PDGF). These factors cause activation ofendothelial cells, promoting the growth of new blood vessels. Sincetumors require a vascular supply to grow, the inhibition of tumor growthby anti-angiogenic drugs has long been identified as an important targetfor research and approach to treatment and has spurred the developmentof several anti-angiogenic agents (AAA).

The first FDA-approved AAA, bevacizumab, is a monoclonal antibody thattargets circulating VEGF A and has been approved for the treatment ofnumerous cancer types, including for example metastatic colorectalcancer, non-small cell lung cancer, kidney cancer andrecurrent-progressive glioblastoma (as monotherapy) or for some of thesame as well as additional cancers in combination with chemotherapeuticdrugs.

Despite high early promise, the addition of anti-angiogenics toconventional chemotherapy drugs has had limited success. In fact,bevacizumab was originally approved for breast cancer but that approvalwas eventually withdrawn for lack of effectiveness. Thus, additionalstudy of the detailed mechanism of anti-angiogenic response, and of thereasons for its failure, is needed in order to more effectively harnessAAAs as a therapeutic modality. Additionally, development and validationare needed of biomarkers suitable for monitoring administration andeffectiveness of AAAs as well as for determining improved dosage andadministration regimen of the chemotherapy arm of combination therapies.

Rao, S. S. et al, Radiotherapy and Oncology 111 (2014) 88-93 (2014)(available online 29 Apr. 2014 incorporated by reference) reports thatthe short-acting AAA axitinib administered shortly before single doseradiation therapy (SDRT) increases acute tumor endothelial cellapoptosis and increases tumor response compared to SDRT administeredalone. Rao specifically reports that the radiosensitization is dependenton the relative timing of administration of the two modalities with theoptimum time being axitinib preceding SDRT by one hour in mice andproducing no significant additive effect when this particularanti-angiogenic agent is administered 2 or more hours earlier than SDRT,or when administered at a point subsequent to SDRT. The authors drawparallels between previously reported anti-angiogenic de-repression ofacid sphingomyelinase driven radiosensitization using anti-VEGF andanti-VEGFR2 antibodies and the radiosensitization observed by use ofaxitinib in combination with SDRT.

However, prior to the work described in this disclosure, the foregoingarticle had no implications for chemotherapy as the variouschemotherapeutic agents have very different mechanisms of action asoutlined above. Moreover, unlike the SDRT response which is known to bemediated in significant part by the endothelial cell ASMase/ceramidepathway, ceramide-mediated endothelial apoptosis has not been reportedor proposed for chemotherapy. (Nor has acute ceramide-mediatedvasoconstriction leading to ischemia reperfusion injury been previouslyproposed for RT or for chemotherapy.)

Dietrich, J et al, Expert Opin Investig Drugs. October 2009; 18(10):1549-1557 doi: 10.1517/13543780903183528 review the use of cediranib, ashort-acting anti-angiogenic agent in the treatment of glioblastoma. Theauthors generally rate the use of cediranib as promising for treatmentof glioblastoma but note the absence of biomarkers for anti-angiogenictherapies. Further, the authors note the phenomenon of transientvascular normalization that follows treatment with anti-angiogenicagents and suggest that a specific treatment window might exist (relatedto vascular normalization) during which chemotherapy and radiation maybe most effective. However, vascular normalization is a relativelyslow-developing event, taking days to become manifest and thus thispaper does not point to a second modality treatment window developingvery close to AAA administration. Lastly, the authors comment that thereasons for re-establishment of pathologic vascularization (after thetransient normalization stage) are poorly understood but if they can beelucidated may offer an explanation for failure of treatments withanti-angiogenic factors precipitated by up-regulation of alternatepro-angiogenic factors which are not targets of the administeredanti-angiogenic agent.

As illustrated by the foregoing, anti-angiogenic drugs have not beensuccessful and an acute need exists to find methods for increasing theircontribution to clinical outcome. Moreover, a general need exists toimprove clinical outcomes in cancer therapy in general.

SUMMARY OF THE DISCLOSURE

Disclosed is a method for enhancing the tumor response to chemotherapycomprising: (a) administering a short-acting anti-angiogenic agent (AAA)to a subject afflicted with a solid tumor, thereby creating a timeinterval of increased susceptibility of said tumor to at least onechemotherapeutic agent; (b) administering said at least onechemotherapeutic agent that has the property of activating theASMase/ceramide signaling pathway to said subject at a time point withinsaid interval; hereby enhancing the effect of the at least onechemotherapeutic agent against said tumor. Enhancement can be assessedby comparison to the chemotherapeutic agent being used (i) without theanti-angiogenic agent or (ii) at a time point outside the interval ofincreased susceptibility (chemosensitization interval).

In some embodiments the foregoing interval has been predetermined for apopulation of subjects or for a particular subject. In some embodiments,the interval has been determined by using IVIM DW-MRI, or by direct orindirect measurements of ASMase activity (for example by measurement ofsurrogates thereof) to determine an interval of acute increase followingan administration of AAA, wherein the interval of acute ASMase activityincrease is the interval of increased susceptibility of the subject tochemotherapy.

In some embodiments, the amount of the anti-angiogenic agent iseffective to cause a substantial increase in ASMase activity in thesubject during said interval. In some embodiments, ASMase activity isassessed by dynamic intravoxel incoherent motion (IVIM)-baseddiffusion-weighted magnetic resonance imaging (DW-MRI). In someembodiments, ASMase activity is assessed by measuring ASMase activity,or deducing ASMase activity by measuring a surrogate, such as one ormore pro-apoptotic ceramides. Suitable pro-apoptotic ceramides includeC16:0 ceramide and C18:0 ceramide.

In some embodiments, the amount of anti-angiogenic agent administered isone causing maximal ASMase activity increase provided that such amountdoes not exceed a maximum tolerated dose of the AAA.

In some embodiments, following a first administration of AAA and said atleast one chemotherapeutic agent, the subject is administered a seconddose of AAA after the lapse of a time interval at least sufficient forthe AAA from the first administration to decay to an extent sufficientfor the responsiveness to ASMase in said subject to a secondadministration of AAA to be reset (reestablishing ASMase sensitivity).The second administration of AAA is followed by a second administrationof said at least one chemotherapeutic agent within an interval ofincreased susceptibility of said tumor created by the secondadministration of AAA. Any subsequent administration of AAA andchemotherapeutic will follow the method of the second administration.

In some embodiments, the tumor is selected from the group consisting ofadrenal (e.g., adrenocortical carcinoma), anal, bile duct, bladder, bone(e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma),brain/CNS (e.g., astrocytoma, glioma, glioblastoma, childhood tumors,such as atypical teratoid/rhabdoid tumor, germ cell tumor, embryonaltumor, ependymoma), breast (including without limitation ductalcarcinoma in situ, carcinoma, cervical, colon/rectum, endometrial,esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder,gastrointestinal, kidney (e.g., renal cell, Wilms' tumor), heart, headand neck, laryngeal and hypopharyngeal, liver, lung, oral (e.g., lip,mouth, salivary gland) mesothelioma, nasopharyngeal, neuroblastoma,ovarian, pancreatic, peritoneal, pituitary, prostate, retinoblastoma,rhabdomyosarcoma, salivary gland, sarcoma (e.g., Kaposi's sarcoma), skin(e.g., squamous cell carcinoma, basal cell carcinoma, melanoma), smallintestine, stomach, soft tissue sarcoma (such as fibrosarcoma),rhabdomyosarcoma, testicular, thymus, thyroid, parathyroid, uterine(including without limitation endometrial, fallopian tube), and vaginaltumor and the metastasis thereof. In some embodiments, the tumor isselected from the group consisting of breast, lung, GI tract, skin, andsoft tissue tumors. In some further embodiments the tumor is selectedfrom the group consisting of breast, lung, GI tract and prostate tumors.

In some embodiments, the short-acting AAA is at least one AAA selectedfrom the group consisting of cediranib, axitinib, anginex, sunitinib,sorafenib, pazopanib, vatalanib, cabozantinib, ponatinib, lenvatinib,and SU6668. In some embodiments the short-acting AAA has an averagehalf-life of up to about 120 hours. Cetuximab is not considered shortacting AAA.

In some embodiments, suitable classes of chemotherapeutic agentsinclude, but not limited to taxanes, DNA alkylating agents,topoisomerase inhibitors, endoplasmic reticulum stress inducing agents,anti-tumor antibiotics, and antimetabolites.

In some embodiments, the chemotherapeutic agent is selected from thegroup consisting of chlorambucil, cyclophosphamide, ifosfamide,melphalan, streptozocin, carmustine, lomustine, bendamustine,uramustine, estramustine, carmustine, nimustine, ranimustine,mannosulfan busulfan, dacarbazine, temozolomide, thiotepa, altretamine,5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea,methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin,idarubicin, SN-38, ARC, NPC, campothecin, topotecan,9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan,diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide,etoposide phosphate, teniposide, doxorubicin, paclitaxel, docetaxel,gemcitabine, baccatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol,cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatinIII, 10-deacetyl cephaolmannine, and mixtures thereof.

In some embodiments, the chemotherapeutic agent is selected from thegroup consisting of gemcitabine, paclitaxel, docetaxel, and etoposide,and mixtures thereof.

In some embodiments, the chemotherapeutic agent is a taxane.

In some embodiments the chemotherapeutic agent is selected from thegroup consisting of baccatin III, 10-deacetyltaxol,7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol,7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, andmixtures thereof.

In some embodiments the chemotherapeutic agent is a DNA alkylatingagent. In further embodiments the chemotherapeutic agent is selectedfrom the group consisting of nitrogen mustards, nitrosoureas, andalkylsulfonates.

In some embodiments the chemotherapeutic agent is selected from thegroup consisting of cyclophosphamide, chlorambucil, melphalan,bendamustine, uramustine, estramustine, carmustine, lomustine,nimustine, ranimustine, streptozotocin; busulfan, mannosulfan, andmixtures thereof.

In some embodiments the chemotherapeutic agent is a topoisomerase Iinhibitor. In further embodiments the chemotherapeutic agent is selectedfrom the group consisting of SN-38, ARC, NPC, camptothecin, topotecan,9-nitrocamptothecin, exatecan, lurtotecan, lamellarinD9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927,DX-8951f, MAG-CPT, and mixtures thereof.

In some embodiments the chemotherapeutic agent is a topoisomerase IIinhibitor. In further he chemotherapeutic agent is selected from thegroup consisting of amsacrine, etoposide, etoposide phosphate,teniposide, daunorubicin, mitoxantrone, amsacrine, ellipticines,aurintricarboxylic acid, doxorubicin, and HU-33 land combinationsthereof.

In some embodiments the chemotherapeutic agent is an antimetabolite. Infurther embodiments the chemotherapeutic agent is selected from thegroup consisting of 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,hydroxyurea, methotrexate, pemetrexed, and mixtures thereof.

In some embodiments the tumor is selected from the group consisting ofadrenal, anal, bile duct, bladder, bone, brain/CNS, breast, cervical,colon/rectum, endometrial, esophageal, eye, gallbladder,gastrointestinal, kidney, heart, head and neck, laryngeal andhypopharyngeal, liver, lung, oral mesothelioma, nasopharyngeal,neuroblastoma, ovarian, pancreatic, peritoneal, pituitary, prostate,retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin, smallintestine, stomach, soft tissue sarcoma, rhabdomyosarcoma, testicular,thymus, thyroid, parathyroid, uterine, and vaginal tumors and metastasesthereof. In further embodiments the tumor is selected from the groupconsisting of breast, lung, GI tract and prostate tumors.

In some embodiments the AAA has a half-life of less than about 120hours, less than about 110 hours, less than about 100 hours, less thanabout 90 hours, less than about 80 hours, less than about 70 hours, lessthan about 60 hours, less than about 50 hours, less than about 40 hours,less than about 35 hours, less than about 30 hours, less than about 25hours, less than about 20 hours, less than about 18 hours, less thanabout 15 hours, less than about 12 hours, less than about 10 hours, orless than about 8 hours. In some embodiments the AAA has a decay periodthat is about the same as the half-life of the AAA.

In some embodiments the AAA is administered at the maximum tolerateddose of the AAA, about 90% of the tolerated dose of the AAA, about 80%of the tolerated dose of the AAA, about 70% of the tolerated dose of theAAA, about 60% of the tolerated dose of the AAA, about 50% of thetolerated dose of the AAA, about 40% of the tolerated dose of the AAA,about 30% of the tolerated dose of the AAA, about 20% of the tolerateddose of the AAA, or about 10% of the tolerated dose of the AAA.

In some embodiments the AAA is administered at the approved dose fordaily administration, about twice the approved dose for dailyadministration, about three times the approved dose for dailyadministration, about four times the approved dose for dailyadministration, about five times the approved dose for dailyadministration, about six times the approved dose for dailyadministration, about seven times the approved dose for dailyadministration, about eight times the approved dose for dailyadministration, about nine times the approved dose for dailyadministration, or about ten times the approved dose for dailyadministration.

In some embodiments the chemotherapeutic agent is administered about 0.5to 5 hours, about 0.5 to 4 hours, about 0.5 to 3 hours, about 0.5 to 2hours, about 0.5 to 1.5 hours, about 0.5 to 1 hour, 1 to 5 hours, about1 to 4 hours, about 1 to 3 hours, about 1 to 2 hours, about 1 to 1.5hour, 1.5 to 5 hours, about 1.5 to 4 hours, about 1.5 to 3 hours, about1.5 to 2 hours, 2 to 5 hours, about 2 to 4 hours, about 2 to 3 hours,about 3 to 5 hours, about 3 to 4 hours, or about 4 to 5 hours afteradministration of the AAA.

In further embodiments the chemotherapeutic agent is administered nomore than about 2 hours or no more than about 1.5 hours or no more thanabout 1 hour after administration of the AAA or the administration ofthe chemotherapeutic agent is commenced within a half-hour afteradministration of the AAA.

Disclosed is also a method for predicting tumor response or monitoringefficacy or timing of treatment of a patient to a chemotherapeuticagent, the patient being afflicted with a malignant solid tumor, themethod comprising: using diffusion-weighted magnetic resonance imaging(DW-MRI) and in more particular embodiments dynamic intravoxelincoherent motion (IVIM)-based diffusion-weighted magnetic resonanceimaging (DW-MRI) shortly following administration of thechemotherapeutic agent to determine the extent of rapid perfusionalterations in the tumor vasculature following administration of thechemotherapeutic agent, wherein an increased amount of alterations overbaseline is indicative of tumor response to said chemotherapeutic agentor of appropriate timing and/or efficacy of treatment.

Other aspects of the present disclosure are directed to:

-   -   a. Methods for predicting tumor response or monitoring timing        and/or efficacy of treatment in a patient afflicted with a        malignant tumor to a chemotherapeutic agent, comprising:        -   measuring an ASMase level or activity in the patient            following administration of the chemotherapeutic agent to            the patient wherein an increase in said level or activity            compared to baseline is indicative of tumor response to the            chemotherapeutic agent or confirms the efficacy and/or            appropriate timing of treatment (and wherein, conversely, an            absence of such decrease carries no such prediction); and    -   b. Methods for predicting tumor response or monitoring timing of        treatment in a patient to a chemotherapeutic agent, the patient        being afflicted with a malignant solid tumor, the method        comprising:        -   using diffusion-weighted magnetic resonance imaging (DW-MRI)            and preferably dynamic intravoxel incoherent motion            (IVIM)-based DW-MRI to measure perfusion alterations            following administration of the chemotherapeutic agent to            determine the extent of rapid perfusion defects in the tumor            vasculature following administration of the chemotherapeutic            agent, wherein an increased level of said alterations over            baseline is indicative of tumor response to said            chemotherapeutic agent or of efficacy or appropriate timing            of treatment (and wherein, conversely, an absence of such            perfusion alterations carries no such indication).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are plots, respectively of ASMase activity (nmols/mg/hr)and ceramide levels (pmols/10⁶ cells) in BAEC against time aftertreatment of endothelial cells with a chemotherapeutic agent, paclitaxel(100 nM).

FIG. 1C is a series of microphotographs showing the formation of CRMover time in BAEC monolayers exposed to paclitaxel (100 nM), in theabsence (upper panel) or presence (lower panel) of 30 μg/mL nystatin (30min pre-treatment). BAEC were co-stained with anti-ceramide antibody(which binds to the cell membrane as indicated by the grey area betweenthe cell nuclei most prominent in the upper right three panels of FIG.1C, wherein the cell nuclei appear as discreet the latter appearing asdiscrete globules and DAPI (to stain nuclei which appear as globules) inorder to localize CRMs to plasma membranes.

FIGS. 1D and 1E are plots, respectively of ASMase activity (nmols/mg/hr)and ceramide levels (pmols/10⁶ cells) in BAEC, against time aftertreatment of bovine endothelial cells with the chemotherapeutic agentetoposide (50 FIG. 1F is a bar graph showing ceramide levels (pmols/10⁶cells) over time (min) in HCAEC treated with 50 μM etoposide.

FIGS. 2A and 2B are plots of incidence of apoptosis (% apoptosis)against time (hrs) in BAEC treated with paclitaxel (100 nM) (FIG. 2A) oretoposide (50 μM) (FIG. 2B). FIG. 2C is a bar graph showing incidence ofapoptosis (% apoptosis) at increasing doses of cisplatin in BAEC.

FIG. 3 is a bar graph showing incidence of apoptosis (% apoptosis) inBAEC pre-incubated with bFGF (2 ng/mL), VEGF (2 ng/mL) or nystatin (30μg/mL) prior to treatment with etoposide (50% apoptosis was determinedwas evaluated after 8 hours of etoposide treatment.

FIG. 4A is a panel of representative 5-μm histologic tumor sectionsobtained either from controls (left panel) or at 4 hours after exposureof tumor-bearing mice to a single dose of paclitaxel (25 mg/kg i.p.,right panel), and stained for endothelial surface marker MECA-32 andTUNEL. FIG. 4B is a bar graph showing quantification of endothelial cellapoptosis after single dose of paclitaxel. FIG. 4C is a panel ofhistologic MCA/129 fibrosarcoma sections obtained either from untreatedcontrols (left panel) or at 4 hours after exposure to single dose ofgemcitabine (240 mg/kg i.p., right panel), and stained for endothelialsurface marker MECA-32 and TUNEL. FIG. 4D is quantification ofendothelial cell apoptosis in MCA/129 tumors at 4 hours after treatmentas in FIG. 4C.

FIGS. 5A and 5B are plots of tumor volume (mm³) versus days post tumorimplantation. FIG. 5A shows tumor volume over time in SCID^(asmase+/+)or SCID^(asmase−/−) mice harboring HCT116 tumors (50-70 mm³) and treatedwith paclitaxel (15/20/25 mg/kg i.p.) three times biweekly. Arrowsindicate days of paclitaxel treatment. Data (mean±SD) are collated from5 mice per group. FIG. 5B shows tumor volume over time inSCID^(asmase+/+) mice harboring HCT116 tumors (50-70 mm³) and treatedwith etoposide (35/35/50 mg/kg i.p.) biweekly in the presence ofanti-ceramide or isotype control antibody. Arrows indicate days ofetoposide treatment. Data (mean±SD) are collated from 5 mice per group.

FIGS. 6A, 6B, and 6C are plots of HCT116 tumor volume (mm³) inSCID^(asmase+/+) mice versus days post tumor implantation. FIG. 6A showsanti-angiogenic chemosensitization of HCT116 tumors in SCIDasmase^(+/+)mice treated with DC101 (1.6 mg per mouse i.v.) 1 hour prior topaclitaxel treatment. FIGS. 6B and 6C show effect of timing of DC101relative to paclitaxel on the response of HCT116 tumors. DC101 (800μg/25 gm mouse i.v.) was provided either before (FIGS. 6B and 6C) orafter (FIG. 6C) paclitaxel treatment. FIG. 6D is a bar graph showingpercent of endothelial cell apoptosis following the administration ofDC101 (1.6 mg/25 gm mouse i.p.) in combination with gemcitabine (240mg/kg i.p.) to MCA/129 tumors implanted in sv129/Bl6^(asmase+/+) mice.FIG. 6E shows the impact of timing of DC101 relative to gemcitabine onthe response of MCA/129 tumors. Experiments were performed as in (FIG.6D) with DC101 timed either 1 hour before or immediately preceding eachdose of gemcitabine (240 mg/kg i.p.). Data (mean±SEM) are collated from5 mice/group. FIG. 6F is a plot of HCT116 tumor volume versus days posttumor implantation in mice treated with various doses of cisplatin.

FIG. 7A includes demographics of patients with metastatic sarcomatreated with bevacizumab, gemcitabine, and docetaxel on a prospectivephase II study at MSKCC. FIG. 7B shows representative CT images of anabdominal wall metastasis at baseline (left), 3 months (center) and 6months (right) in patient #8 in the 1-hour interval bevacizumab cohort(FIG. 7D). FIGS. 7C and 7D are waterfall plots demonstrating best tumorresponse based on volumetric change for each patient treated withbevacizumab immediately before (FIG. 7C) or 1 hour before (FIG. 7D)gemcitabine. FIGS. 7E and 7F are the same types of plots as 7C and 7Dfor the same patients after two cycles of treatment with bevacizumab andgemcitabine administered as for FIGS. 7C and 7D.

FIG. 8 is a bar graph of fold change in serum ceramide (C16:0 and C18:0)levels in MCA129 fibrosarcoma tumor allografts treated with 27 Gy IR at24 hours post-irradiation (24 h).

FIG. 9 are plots that demonstrate SDRT induced microvascular changes inpatients. FIG. 9A shows two values obtained by IVIM DW-MRI representingthe fraction of moving blood (f) and the velocity of blood (D*) in themicrocirculation. Shown is a schematic representation of the calculationof f and D* from the biexponential decay diffusion curve. FIGS. 9B and9D are dot plots showing a baseline, pre-SDRT D* and f values for allpatients in each patient cohort. FIG. 9C and FIG. 9E are dot plotsshowing fold changes in f and D* after RT. Each point represents onepatient with value expressed as the f or D* value as a fraction of thatpatient's pre-radiation value. Up to 16 repeated measurements of D* andf were used to determine the pre- and post-SDRT values. Mean andstandard deviations of the 9 Gy and 24 Gy cohorts are shown. Two-tailed,unpaired t tests with Welch's correction were used in FIGs D and E.

FIG. 10 shows that serum ASMase activity changes in patients receivingSDRT in a dose dependent manner. FIG. 10A is a graph showing normalizedfold changes in ASMase activity 24 hours after 9 or 24 Gy. Three datapoints are represented for each patient: 0(pre-RT), 1 hour and 24 hourspost RT. Error bars represent the standard error of 3 technicalreplicates of ASMase measurements for each sample. FIG. 10B is a bargraph showing mean fold changes in serum ASMase activity in the twopatient cohorts. Two tailed, unpaired t tests in B. *P<0.05. FIG. 10Cshows age, gender and tumor type for patients in each cohort.

FIG. 11A is a plot of MCA/129 fibrosarcoma tumor volume versus days posttumor implantation showing that long acting anti-angiogenic drugs (suchas DC101) render tumors refractory to subsequent anti-angiogenicASMase-mediated chemotherapy chemosensitization. Data (mean±SEM) werecollated from 5 mice/group. FIG. 11B is a series of plots of MCA/129fibrosarcoma tumor volume versus days post tumor implantation, where thedata depict the individual tumor response profile collated in FIG. 11A.

FIG. 12A is a series of graphs showing tumor profiles (tumor volume overdays post tumor implantation). Complete responses, defined as nopalpable tumor at 30 days post-IR, are summarized in FIG. 12B. FIG. 12Cis a bar graph depicting a relationship between radiation dose andASMase activity changes 24 hours following IR. FIG. 12D shows acorrelation between change in ASMase activity 24 hours post-IR, andtumor control. Number of mice is indicated in parentheses in 12B and12C. *P<0.05, **P<0.01, ***P<0.001. Two tailed, unpaired, student's ttest was used in 12C. Pearson's Correlation Coefficient was used in 12D.

FIG. 13 indicates that changes in ASMase activity predict tumorresponses to SDRT. FIG. 13A shows changes in ASMase activity 24 hourspost-IR (27 Gy in each mouse (total of 19 animals), ranked by inducedactivity. Means (±SE) of 3 serum measurements are displayed. FIG. 13B isa bar graph indicating a relationship between induced ASMase activityand ultimate tumor responses. Two tailed, unpaired t tests were used inFIG. 13B. *P<0.05, **P<0.01, ***P<0.001.

FIG. 14A shows that gemcitabine rapidly reduces vascular perfusion infibrosarcoma tumors and that gemcitabine activates ASMase in a dosedependent manner. FIG. 14A is a graph of vascularity (Fp) over time infibrosarcoma tumors. FIG. 14B is a graph of diffusion over time infibrosarcoma tumors. FIG. 14C is a graph of ASMase activity over timefollowing the treatment of cultured bovine aortic endothelial cells with100 nM gemcitabine. FIG. 14D is a graph of ASMase activity following thetreatment of bovine aortic endothelial cells with increasing doses ofgemcitabine (measurements taken at 5 minutes following the treatment).

DETAILED DESCRIPTION

The present disclosure is based on the following discoveries:

-   -   The ASMase/ceramide pathway is activated in endothelial cells by        chemotherapeutic agents that belong to distinct classes of        chemotherapeutic agents, such as: taxanes, topoisomerase        inhibitors, and nucleoside analog metabolic inhibitors.        Paclitaxel, etoposide and gemcitabine were shown to activate        ASMase signaling and generation of ceramide in cultured bovine        aortic endothelial cells (BAEC) and human coronary artery        endothelial cells (HCAEC). Activation of ASMase/ceramide pathway        leads to the formation of ceramide-rich microdomains (CRMs).        Cisplatin failed to elicit apoptosis in BAEC but it is known to        induce apoptosis and to increase ASMase in human cells (Lacour        et al. Cancer Res. 15; 64(10):3593-8 (2004); Maurmann et al.        Apoptosis, 20:7, 960-974 (2015). Based on this, cisplatin        therapy is also expected to take advantage of chemosensitization        according to the present disclosure.    -   1. Paclitaxel, etoposide and gemcitabine also trigger        endothelial dysfunction, as evident by endothelial apoptosis        both in vitro and in vivo. Cisplatin also induces apoptosis in        human endothelial cells.    -   2. Endothelial apoptosis is inhibited by pre-incubation of cells        with pro-angiogenic factors that prevent ASMase activation and        ceramide generation, such as VEGF, bFGF, and nystatin.    -   3. ASMase signaling is required for chemotherapy-induced        apoptotic response in vivo. Furthermore, the t ASMase/ceramide        pathway mediates tumor growth delay caused by chemotherapy        agents that have the ability to activate ASMase/ceramide        signaling.    -   4. Human HCT-116 colon cancer and MCA/129 murine sarcoma        xenografts implanted in wild-type asmase^(+/+) mice undergo        endothelial, but not tumor cell, apoptosis following the        treatment with chemotherapeutic agent paclitaxel or etoposide.        Additionally, chemotherapy treatment results in significant        tumor growth delay in wild-type asmase^(+/+) mice. The opposite        is observed when xenografts are implanted in asmase^(−/−) mice.        Tumors generated in mice deficient in ASMase signaling show lack        of endothelial cell apoptosis and no significant tumor response        when treated with paclitaxel or etoposide. Finally, intravenous        injection of anti-ceramide IgM 1 hour prior to administration of        chemotherapeutic agents abrogates or attenuates the benefits of        chemotherapy on tumor growth in these models. Collectively,        these results indicate that ASMase signaling and ceramide        formation are required for chemotherapy-induced endothelial cell        apoptosis and optimal tumor response.    -   5. Anti-VEGFR2 antibody DC101, which de-represses endothelial        ASMase inhibited by VEGF, shows a synergistic tumor response        when used in combination with paclitaxel or gemcitabine in        HCT116 tumors and MCA/129 fibrosarcomas, but only when        administered 1-2 hours prior to, and not immediately preceding,        chemotherapy treatment. However, the synergistic effect of        anti-angiogenic agent and chemotherapy is completely abolished        when HCT-116 and MCA/129 xenografts were implanted in host        asmase^(−/−) mice that provide injury-resistant microvasculature        to tumors. Together, these observations indicate the existence        of a previously unreported chemotherapy-induced tumor        microvascular response mechanism that acts synergistically with        chemotherapeutic impact on tumor cells and that is required for        optimal tumor response.    -   6. Anti-angiogenic chemosensitization is contingent on precise        timing of anti-angiogenic delivery, and activity of the        ASMase/ceramide pathway. Chemosensitization occurs only if        anti-angiogenic agent was delivered within the acutely increased        ASMase activity window (chemosensitization window) following        administration of AAA. In the systems of experiments described        here, this window was observed to have a duration of 1-2 hours        preceding chemotherapy, but at no other time preceding or after        chemotherapy. Thus, there is a chemosensitization interval or        window following the administration of anti-angiogenic agent        during which treatment with chemotherapy should occur.    -   7. Synchronized timing of AAA with chemotherapy, where AAA is        delivered prior to the administration of chemotherapy such that        chemotherapy is administered (or at least its administration is        commenced) within the chemosensitization window, significantly        improves tumor response to chemotherapy. Overall, clinical        outcome in the studies presented here represents        proof-of-principle that ASMase signaling can be engaged for        therapeutic benefit. These results also support that ASMase        signaling can be established as a biomarker for predicting tumor        response and in any event for adjusting timing and dosage of the        AAA, optimum timing of the chemotherapeutic drug (relative to        the AAA administration) and dosage of the chemotherapeutic drug.    -   8. Long-acting anti-angiogenic agents (drugs) render tumors        refractory to subsequent anti-angiogenic ASMase-mediated tumor        chemosensitization, where the strength of inhibition intensifies        as the amount of long-acting anti-angiogenic agent present in        the subject increases. Thus, these experiments support the        proposition that short-acting anti-angiogenic drugs are        preferred for ASMase/ceramide pathway-based chemosensitization        compared to long-acting anti-angiogenics such as bevacizumab or        DC101.    -   9. Phase II human clinical trial data show that bevacizumab        delivery (on day zero of a three-week cycle) 1 hour before the        administration of chemotherapy agent gemcitabine in patients        treated for metastatic sarcoma, followed by administration of        docetaxel on day 8 significantly improves tumor response.    -   10. Single dose radiotherapy (SDRT) induces microvascular        vasoconstriction in MCA/129 murine sarcomas and B16 melanomas        implanted in asmase^(+/+) mice following 20 Gy SDRT. On the        contrary, MCA/129 murine sarcomas and B16 melanomas implanted in        asmase null animals do not exhibit perfusion dysfunction        following the same exact treatment. These findings suggest a        causal relationship between ASMase activity and vascular        dysfunction.    -   11. A clinical study including 15 patients with bone metastases        showed that SDRT induces perfusion reduction or ischemia        reperfusion injury (attributed to microvascular        vasoconstriction) following 24 Gy SDRT. These results suggest        that perfusion alterations can be used as a biomarker indicative        of effective treatment. Furthermore, development of such        perfusion alterations as a biomarker can be used to dose        de-escalate SDRT.    -   12. ASMase activity follows the same trend as perfusion        alterations due to microvascular vasoconstriction in patients        treated with 24 Gy. ASMase levels in the serum of 18 patients        increased following the 24 Gy SDRT, suggesting that ASMase        activity can serve as a serum biomarker of clinical success.        These results are consistent with the findings in pre-clinical        animal models, which revealed that perfusion alterations are        dependent on ASMase activity. It is anticipated that a similar        microvascular vasoconstriction manifest as perfusion alterations        (likely developing at a somewhat slower pace) will be observed        if chemotherapy, rather than radiation, is used as a second        therapeutic modality.    -   13. The intensity of ASMase serum activity increase correlates        directly with dose-dependent probability of complete response        after SDRT of MCA/129 fibrosarcoma. These findings indicate that        ASMase levels and/or activity may serve as a serum biomarker in        human cancer management.    -   14. Microvascular vasoconstriction occurs immediately following        administration of a chemotherapeutic agent activating ASMase        signaling in animal model of fibrosarcoma, and Gemcitabine        activates ASMase in a dose-dependent manner. It is anticipated        that other ASMase activating chemotherapeutics will perform        qualitatively the same.    -   15. Ionizing radiation results in translocation of ASMase to the        plasma membrane, generating ceramide and ceramide-rich        platforms. VEGF inhibits ASMase activation through VEGFR2        receptor signaling whereas anti-angiogenics can de-repress        ASMase activation if given 1-2 hours prior to radiation.        Secretion of ASMase into the blood may serve as a biomarker of        upstream ASMase activation. Ceramide-rich platforms lead to        downstream endothelial cell dysfunction, including decreased        tumor microvascular perfusion which can be observed with IVIM        DW-MRI and likely other imaging modalities.    -   16. As shown here, an increase in SDRT dose was directly        proportional to the observed increase in serum ASMase activity,        which in turn directly correlated with percent SDRT-induced        complete tumor response (FIG. 12). Similarly, an increase in        gemcitabine dose proportionally increased ASMase activity (FIG.        14D). These observations indicate that therapeutic targeting of        factors known to inhibit ASMase, such as angiogenic growth        factors (for example VEGF signaling) would lead to an increase        in ASMase activity, which in turn results in enhanced responses        to SDRT or chemotherapy. Thus, use of anti-angiogenic agents        followed by SDRT or chemotherapy delivered within the time        window of acute ASMase activation will increase tumor response        proportional to the intensity of ASMase activation. Hence, it        would be desirable to use the highest dosage of anti-angiogenic        agent feasible and safe, to maximize ASMase activation and the        consequently increased SDRT or chemotherapy response if        delivered within the restricted ASMase-activation time window.        Whereas SDRT and chemotherapy are often administered at or near        their maximum tolerated dose to maximize the anti-tumor effect,        tumor response radiation or chemosensitization by maximal        anti-angiogenic dosage is expected to increase the beneficial        effect of the combined therapy disclosed herein and may even        enable de-escalation of the radiation or chemotherapy dose,        titrated to the radiation or anti-cancer drug dose required to        achieve local and/or systemic tumor cure when used in properly        timed combination with anti-angiogenic therapy.

Based on the foregoing, the present inventors have devised treatmentmodifications to take advantage of these observations and developedbiomarkers for assessing and predicting treatment outcome and monitoringtreatment efficacy and relative timing of administration ofanti-angiogenic agents and chemotherapeutic drugs.

Definitions

As used herein, the following terms and abbreviations shall have themeaning ascribed to them below unless the context clearly indicatesotherwise.

“AAA” means and is used interchangeably with “anti-angiogenic agent.”

“CRM” means “ceramide-rich macrodomain.”

“Decay” in connection with an administered AAA means the inactivation,binding or clearance of such agent in or from the body of a patient suchthat the agent can no longer exert substantial activity.

“Subject” means a patient (human or veterinary) or an experimentalanimal, such as a mouse or other rodent.

“Synergistic effect,” “synergy,” or “synergistic tumor response” meansthe effect of two or more active agents administered as described hereinis greater than the sum of the effects each agent would produce had theagent been administered alone. With specific reference to timing, a“synergistic effect” is a therapeutic effect of two or more activeagents wherein a second-administered agent is administered (or itsadministration is at least commenced) within a chemosensitizationinterval created by administration of the first such that the effect ofthe appropriately timed administration is greater than the effect of aninappropriately timed administration wherein the second administeredtherapeutic agent is administered outside the chemosensitization windowcreated by the first agent.

“Time interval of increased susceptibility” or “chemosensitizationinterval” refers to the period of time after administration of the AAAwherein the tumor response to chemotherapy is increased or whereinASMase activity/ceramide signaling is acutely increased.

“Substantial” with particular reference to an increase in ASMaseactivity, perfusion alteration, increase in ceramide level or othermeasured or derived parameter will mean an increase or other change thatis rapid and clearly observable. For example a change reachingstatistical significance would be considered substantial. Astatistically significant ASMase activity increase in response to achemotherapeutic agent can be qualitatively similar to that of FIG. 14C.Furthermore, ceramide levels (C16:0, C18:0) undergoing a statisticallysignificant increase following chemotherapy would increase in a mannerqualitatively similar to that of FIG. 8.

“Restoration of biologic output” of ASMase or ceramide or any othermeasured parameter refers to a reestablishment of the ability in theendothelium of the host to undergo an increase in ASMase (or otherparameter) activity or expression upon a second administration of AAAafter decay of a first administration of AAA.

Short-Term Acting Anti-Angiogenic Agents (AAA)

Anti-angiogenic agents (AAA) suitable for use in the present method areshort-acting AAAs with shorter average decay periods as compared tolong-acting AAAs such as bevacizumab, which has an active half-life inthe body of a patient of approximately three weeks, and DC101. As usedherein, a decayed anti-angiogenic agent shall no longer be available atlevels sufficient to cause ASMase in the patient to become substantiallyrefractory (and consequently not substantially increase in response) toa subsequent AAA treatment. In other words, the decay of the AAA shouldbe sufficient to reset ASMase sensitivity to a new administration ofAAA. The decay period of an AAA is assessed by measuring one or more ofserum levels of said agent, restoration of biologic output of ASMase,restoration of biologic output of ceramide or perfusion alteration.

Short-acting AAAs suitable for use in the present invention haveconsiderably shorter average decay periods measured in hours (up toabout 120 hours). Suitable AAAs include, but are not limited to:cediranib (average plasma half life of about 22 to 27 hours and a peakplasma concentration of 2-8 hours after administration), axitinib(average half-life 2.5 to 6 h), anginex (half-life ˜50 minutes),sunitinib (average half-life of 40-60 hours), sorafenib (averagehalf-life of about 25-48 hours), pazopanib (average half-life of about30 hours), vatalanib (average half-life of 4.7 hours), cabozantinib(average half-life of 55 hours), ponatinib (average half-life of 24hours); lenvatinib (average half-life of 28 hours) and SU6668 (averagehalf-life of 3.6 hours). Half-life is an indicator of decay periodalthough decay (such that a residual amount remaining from a firstadministration will not be enough to foreclose ASMase activation upon asubsequent administration of AAA) is also dependent on the doseadministered.

In certain embodiments of the invention, the AAA has a decay period ofless than about 120 hours, less than about 110 hours, less than about100 hours, less than about 90 hours, less than about 80 hours, less thanabout 70 hours, less than about 60 hours, less than about 50 hours, lessthan about 40 hours, less than about 35 hours, less than about 30 hours,less than about 25 hours, less than about 20 hours, less than about 18hours, less than about 15 hours, less than about 12 hours, less thanabout 10 hours, or less than about 8 hours.

In further embodiments of the invention, the AAA has a decay period fromabout 1 to 3 hours, from about 1 to 5 hours, from about 1 to 7 hours,from about 1 to 10 hours, from about 1 to 15 hours, from about 1 to 20hours from about 1 to 25 hours, from about 1 to 30 hours, from about 1to 40 hours, from about 1 to 50 hours, from about 1 to 60 hours fromabout 1 to 70 hours, from about 1 to 80 hours, from about 1 to 90 hours,from about 1 to 100 hours, from about 2 to 3 hours, from about 2 to 5hours, from about 2 to 7 hours, from about 2 to 10 hours, from about 2to 15 hours, from about 2 to 20 hours from about 2 to 25 hours, fromabout 3 to 5 hours, from about 3 to 7 hours, from about 3 to 10 hours,from about 3 to 15 hours, from about 3 to 20 hours from about 3 to 25hours, from about 3 to 30 hours, from about 5 to 7 hours, from about 5to 10 hours, from about 5 to 12 hours, from about 5 to 15 hours, fromabout 5 to 20 hours from about 5 to 25 hours, from about 5 to 30 hours,about 5 to 40 hours, from about 5 to 50 hours, from about 5 to 60 hoursfrom about 5 to 70 hours, from about 5 to 80 hours, from about 5 to 90hours, from about 5 to 100 hours, from about 7 to 10 hours, from about 7to 12 hours, from about 7 to 15 hours, from about 7 to 20 hours fromabout 7 to 25 hours, from about 7 to 30 hours, from about 7 to 35 hours,from about 7 to 40 hours, from about 10 to 12 hours, from about 10 to 15hours, from about 10 to 20 hours, from about 10 to 25 hours, from about10 to 30 hours, about 10 to 40 hours, from about 10 to 50 hours, fromabout 10 to 60 hours from about 10 to 70 hours, from about 10 to 80hours, from about 10 to 90 hours, from about 10 to 100 hours, from about20 to 25 hours, from about 20 to 30 hours, about 20 to 40 hours, fromabout 20 to 50 hours, from about 20 to 60 hours from about 20 to 70hours, from about 20 to 80 hours, from about 20 to 90 hours, or fromabout 20 to 100 hours.

It is an important aspect of the present disclosure that short-actingAAA's will be advantageous for use in the timed combination of AAAadministration and chemotherapy such that the AAA sensitizes the patientto chemotherapy and wears off before a subsequent dose of AAA is given.If the prior dose has not worn off, as can be the case with, e.g.,bevacizumab or DC101, the ASMase signaling of the patient becomesrefractory and no longer responds to a fresh dose of AAA.

Another important aspect of this disclosure is that, based on theshowing by the inventors that ASMase activation correlates with survivalof subjects including human patients, the optimum amount of short-actingAAA administered is anticipated to be several times higher than thecurrently approved dose designed for daily administration and may be ashigh as the maximum tolerated dose of AAA. This amount is of coursesubject to optimization, which can be achieved using the teachingsprovided herein to achieve maximal ASMase activation (subject to anydose-limiting toxicity).

Chemotherapeutic Agents

Chemotherapeutic agents suitable for use in the methods and usesdescribed below include agents that have the property of activatingASMase/ceramide signaling in a patient (for example, in endothelialcells). As is shown below, representatives of several but not allclasses of chemotherapeutic agents have this property. In any event, aprocedure for ascertaining this property is described herein. See, forexample below, Materials and Methods and Example 1.

In certain embodiments, suitable chemotherapeutic agents include, butare not limited to taxanes, topoisomerase inhibitors and antimetabolites(e.g., nucleoside analogs acting as such, for example, gemcitabine).Other classes of chemotherapeutics may be included contingent on theirpossessing the property of activating ASMase. Nonlimiting examples to beconsidered include alkylating agents, antimetabolites, anti-tumorantibiotics, mitotic inhibitors, and other chemotherapeutic agentscapable of activating ASMase/ceramide signaling pathway.

In certain embodiments, suitable chemotherapeutic agents excludeplatinum containing chemotherapeutics. In further embodiments of theinvention, suitable chemotherapeutic agents exclude cisplatin.

In still further embodiments, more than one chemotherapeutic agent maybe administered. In certain embodiments, a first chemotherapeutic agentmay be administered within the chemosensitization interval of the AAAand a second chemotherapeutic agent may be administered after thiswindow. In other embodiments, more than one chemotherapeutic agent maybe administered within the chemosensitization interval of the AAA. Forexample, in Example 21, both gemcitabine and docetaxel would beadministered although only one, gemcitabine, is administered during theincreased ASMase activity or signaling interval or window. However, ifco-administration or immediately sequential administration of a secondchemotherapeutic is indicated, such administration may be advantageouslyeffected during the increased ASMase signaling interval. Alternatively,if the process is repeated such that administration of AAA is repeatedafter substantial decay of the AAA in the subject's body (and thesensitivity to AAA-induced acute ASMase activation is reset). The second(and any subsequent) AAA administration creates a new increased ASMasewindow. The chemotherapeutic can then be administered during that windowof chemosensitization and if a second chemotherapeutic agent isindicated, it can be administered substantially simultaneously withinthe same window of chemosensitization or in alternating cycles withinsuccessive windows of chemosensitization), with administration of achemotherapeutic agent always following closely administration of AAA soas to be within the window.

Thus, based on the findings of the present disclosure, manychemotherapeutic agents and their derivatives and/or functionalanalogues are expected to possess ASMase activating property, including,but not limited to taxanes, DNA alkylating agents, topoisomeraseinhibitors, endoplasmic reticulum stress inducing agents, anti-tumorantibiotics, antimetabolites, etc.

In some embodiments, the chemotherapeutic agent is selected from thegroup consisting of chlorambucil, cyclophosphamide, ifosfamide,melphalan, streptozocin, carmustine, lomustine, bendamustine,uramustine, estramustine, carmustine, nimustine, ranimustine,mannosulfan busulfan, dacarbazine, temozolomide, thiotepa, altretamine,5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea,methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin,idarubicin, SN-38, ARC, NPC, campothecin, topotecan,9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan,diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide,etoposide phosphate, teniposide, doxorubicin, paclitaxel, docetaxel,gemcitabine, baccatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol,cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatinIII, 10-deacetyl cephalomannine, and combinations thereof.

In some embodiments, the chemotherapeutic agent is selected from thegroup consisting of gemcitabine, paclitaxel, docetaxel, and etoposide,and combinations thereof.

In particular embodiments, the methods described herein can be used inthe treatment of various types of solid tumors. Examples of solid tumorsinclude, but are not limited to tumors of the following organs: theskin, breast, brain, cervix, testis, heart, lung, gastrointestinaltract, genitourinary tract, liver, bone, nervous system, reproductivesystem, and adrenal glands.

Malignant tumors which can be treated by methods described herein can beused in the treatment of cancer, include without limitation adrenaltumors (e.g., adrenocortical carcinoma), anal, bile duct, bladder, bonetumors (e.g., Ewing's sarcoma, osteosarcoma, malignant fibroushistiocytoma), brain/CNS (tumors e.g., astrocytoma, glioma,glioblastoma, childhood tumors, such as atypical teratoid/rhabdoidtumor, germ cell tumor, embryonal tumor, ependymoma), breast tumors(including without limitation ductal carcinoma in situ, carcinoma,cervical, colon/rectum, endometrial, esophageal, eye (e.g., melanoma,retinoblastoma), gallbladder, gastrointestinal, kidney (e.g., renalcell, Wilms' tumor), heart, head and neck, laryngeal and hypopharyngeal,liver, lung, oral (e.g., lip, mouth, salivary gland) mesothelioma,nasopharyngeal, neuroblastoma, ovarian, pancreatic, peritoneal,pituitary, prostate, retinoblastoma, rhabdomyosarcoma, salivary gland,sarcoma (e.g., Kaposi's sarcoma), skin (e.g., squamous cell carcinoma,basal cell carcinoma, melanoma), small intestine, stomach, soft tissuesarcoma (such as fibrosarcoma), rhabdomyosarcoma, testicular, thymus,thyroid, parathyroid, uterine (including without limitation endometrial,fallopian tube), and vaginal tumor and the metastases thereof. In someembodiments, the tumor is selected from the group consisting of breast,lung, GI tract, skin, and soft tissue tumors.

Dosage, Relative Timing and Administration

AAA Dosage:

As discussed above, in certain embodiments, the AAA dose can be up toseveral times higher than the currently approved dose designed for dailyadministration and could be as high as the maximum tolerated dose ofthat particular AAA. In certain embodiments of the invention, the AAA isadministered at the maximum tolerated dose of the AAA, about 90% of thetolerated dose of the AAA, about 80% of the tolerated dose of the AAA,about 70% of the tolerated dose of the AAA, about 60% of the tolerateddose of the AAA, about 50% of the tolerated dose of the AAA, about 40%of the tolerated dose of the AAA, about 30% of the tolerated dose of theAAA, about 20% of the tolerated dose of the AAA, or about 10% of thetolerated dose of the AAA. In further embodiments of the invention, theAAA is administered at the approved dose for daily administration, abouttwice the approved dose for daily administration, about three times theapproved dose for daily administration, about four times the approveddose for daily administration, about five times the approved dose fordaily administration, about six times the approved dose for dailyadministration, about seven times the approved dose for dailyadministration, about eight times the approved dose for dailyadministration, about nine times the approved dose for dailyadministration, or about ten times the approved dose for dailyadministration. Accordingly the effective dose range of AAA as achemosensitization agent will be broader than that of AAA as currentlyapproved or, if not yet approved, contemplated for therapy.

The dosage of anti-angiogenic agent to be employed forchemosensitization can be ascertained by conducting studies, such asthat described in Example 21. However, if not additional increase inASMase activity (for example by measuring ASMase directly orsphingolipid or ceramide) is observed with an incremental increase inAAA dose, no additional drug should generally be administered asdetailed in Example 21.

The approved dosages of several short-acting AAAs are provided below asadditional guidance. Based on clinical results available and approveddosage information for axitinib, it is anticipated that a dosage rangeof axitinib will exceed 20 mg, up to a dose that induces maximal ASMaseactivation but not higher than a maximum tolerated dose (MTD) will beeffective. 20 mg is the current daily dose used in metastatic renalcancer therapy. Of course, this range is subject to adjustment andoptimization in order to obtain as high ASMase activation as possible.For example, this can be done as outlined in Example 21. Similarly,dosage determination of sunitinib would start with oral administrationof an amount between 2.5 mg and higher up to the MTD mg and escalated insuch increments, proceeding otherwise outlined in the precedingsentences.

TABLE 1 Published Amounts of Short-Acting Anti-angiogenic Agents AAAApproved or Clinically Used Dosage(s) Axitinib Starting dose 5 mg BID(starting dose can be increased or reduced by 1 or 5 mg incrementsSunitinib 37.5/day or 50 mg/day depending on indication (adjustmentsavailable in 12.5 mg increments) Cediranib Not FDA approved: 30 mg/dayhas been used in an NIH clinical study Pazopanib 800 mg/day (iftolerated can be increased) in 200 mg increments

Any suitable method of administration oral or parenteral indicated forthe AAA may be used. In certain embodiments, the AAA is administeredorally. In further embodiments, the AAA is administered parenterally.

Chemotherapeutic Agent Dosage

In certain embodiments of the disclosure, the amount of chemotherapeuticagent will be no higher than a dose-limiting toxicity amount. Forexample, the chemotherapeutic agents provided below are indicated (intheir FDA labels) to be dosed at the following amounts:

Chemotherapeutic Agent Recommended Dosage Paclitaxel 135 mg/m2 to 175mg/m2 (although dosages up to 420 mg/m2 have been used in safetystudies) infused i.v. over 3 or 4 hours (and up to 24 hours) every threeweeks Docetaxel from 60 to 100 mg/m2 with 75 mg/m2 being a commonlyprescribed amount for various indications Etoposide Oral or ivadministration. IV: 5 to 100 mg/m2 every day or every other day for 5days; oral dose is generally 2x times the iv amount. Gemcitabine From1000 mg/m2 to 12000 mg/m2 depending on indication and tolerance (can beadjusted in 200 mg increments)As described in Example 17, ASMase activation occurs in a dose-dependentmanner upon administration of a chemotherapeutic (FIG. 14D).Furthermore, ASMase activation has been shown by the present inventorsto correlate with survival. This indicates that the dose of chemotherapydrug, like the dose of SDRT, should be increased to the point wheremaximal ASMase activity is achieved provided of course that the maximumtolerated dose is not reached earlier.

Frequency and Timing:

The timing of chemotherapy administration should be arranged to fallwithin the increased ASMase activity. In particular embodiments of theinvention, the chemotherapeutic agent is administered about 0.5 to 5hours, about 0.5 to 4 hours, about 0.5 to 3 hours, about 0.5 to 2 hours,about 0.5 to 1.5 hours, about 0.5 to 1 hour, 1 to 5 hours, about 1 to 4hours, about 1 to 3 hours, about 1 to 2 hours, about 1 to 1.5 hour, 1.5to 5 hours, about 1.5 to 4 hours, about 1.5 to 3 hours, about 1.5 to 2hours, 2 to 5 hours, about 2 to 4 hours, about 2 to 3 hours, about 3 to5 hours, about 3 to 4 hours, or about 4 to 5 hours after administrationof the AAA. In certain embodiments of the invention, thechemotherapeutic agent is administered no more than about 2 hours afteradministration of the AAA. In further embodiments of the invention, thechemotherapeutic agent is administered no more than about 1.5 hoursafter administration of the AAA. In still further embodiments of theinvention, the chemotherapeutic agent is administered no more than about1 hour after administration of the AAA. In yet further embodiments theadministration of the chemotherapeutic agent is commenced within ahalf-hour after administration of the AAA. The duration of chemotherapyinfusion will generally be monitored for that which produces the bestsynergy with the AAA, again attempting different rates of infusion,using ASMase signaling (or perfusion alteration) as a biomarker and/ormonitoring tumor response.

In certain embodiments, chemotherapy administration takes place in termsof dosage, frequency, and duration accordance with the indication of theparticular chemotherapeutic agent used. The frequency of administrationwill generally be once on the day of treatment and the space betweentreatments with AAA will be determined taking into account the decaytime of the anti-angiogenic agent as mentioned above and the time periodbetween cycles of the chemotherapy. Typically chemotherapeutic agentsthat are administered parenterally are infused over a period of timeranging from about 20 minutes up to about an hour.

EXAMPLES Materials and Methods: Cell Culture

BAEC were established from the intima of bovine aorta as described¹.Stock cultures were grown in 100-mm dishes in Dulbecco's modifiedEagle's medium (DMEM) supplemented with glucose (1 g/liter), 5%heat-inactivated calf serum (CS), penicillin (50 units/ml), andstreptomycin (50 μg/ml). Purified human recombinant bFGF (1 ng/ml; R&DSystems, Inc., Minneapolis, Minn.) was added every other day during theexponential growth phase. After 8-10 days in culture, cells reachconfluence and exhibit features of contact-inhibited monolayers. Theseplateau phase cells were either used for experiments, or furthersub-cultured (up to a maximum of 10 times) at a split ratio of 1:8. Forsub-culturing, monolayers were dissociated with STV (0.05% trypsin and0.02% EDTA in PBS) for 2-3 min at 22° C., washed twice in 5% CS-DMEM,and resuspended in DMEM with supplements as above. These mild conditionsof trypsinization were sufficient to detach cells but not injure,stimulate, or affect cell function in a detectable way. BAEC weremaintained at 37° C. in 10% CO₂ humidified incubators.

HCAEC were obtained from Clonetics™ Coronary Artery Endothelial CellSystems (Cambrex Bio Science Inc.). For HCAEC culturing andsub-culturing Clonetics cell system components were used: EBM®-2,Endothelial Cell Basal Medium-2 with addition of Clonetics EGM-2-MVSingleQuots containing growth supplements (Cambrex) (BiomedicalTechnologies, Inc.). For sub-culturing, monolayers were dissociated withClonetics® Trypsin/EDTA solution for 2-3 min at 22° C. at a split ratioof 1:4 to expand the cell population for experiments. HCAEC cultureswere maintained at 37° C. in 5% CO₂ humidified incubators.

Apoptosis In Vitro

Apoptosis was assessed in vitro by examining morphologic changes innuclear chromatin using bis-benzimide trihydrochloride (Hoechst #33258;Sigma-Aldrich, Milwaukee Wis.). BAEC and HCAEC monolayer cultures werefirst detached using 0.25% trypsin and 0.02% EDTA in HBSS and thencombined with the floating population of cells. The cell pellet waswashed in PBS, resuspended in 3% paraformaldehyde, and incubated for 10min at 22° C. Following the removal of fixative, cells were resuspendedin PBS containing 8 μg/ml of Hoechst-33258. Following a 15-minincubation at 22° C., cells were placed on a glass slide, and scored forthe incidence of apoptotic chromatin changes using fluorescencemicroscope. Cells exhibiting at least three apoptotic bodies werecounted as apoptotic.

Ceramide Quantitation

After treatment, cells were placed on ice, washed with cold PBS, andlipids were extracted by addition of scraped cells in methanol to anequal volume of chloroform and 0.6 volume of buffered saline/EDTAsolution (135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl₂, 0.5 mM MgCl2, 5.6 mMglucose, 10 mM HEPES pH 7.2, 10 mM EDTA). Lipids in the organic phaseextract were dried under N₂ and subjected to mild alkaline hydrolysis(0.1 N methanolic KOH for 1 hour at 37° C.) to removeglycerophospholipids. Samples were re-extracted, and the organic phasewas dried under N₂. Ceramide contained in each sample was resuspended ina 100 μl reaction mixture containing 150 μg of cardiolipin, 280 μMdiethylenetriaminepentaacetic acid, 51 mM octyl-β-D-glucopyranoside(Calbiochem), 50 mM NaCl, 51 mM imidazole, 1 mM EDTA, 12.5 mM MgCl₂, 2mM dithiothreitol, 0.7% glycerol, 70 μM β-mercaptoethanol, 1 mM ATP, 10μCi of [γ-³²P]ATP, 35 μg/ml Escherichia coli diacylglycerol kinase(Calbiochem) at pH 6.5. After 30 minutes at room temperature, thereaction was stopped by extraction of lipids with 1 ml ofchloroform:methanol:1 N HCl (100:100:1), 170 μl of buffered salinesolution (BSS) (135 mM NaCl, 1.5 mM CaCl₂), 0.5 mM MgCl2, 5.6 mMglucose, and 10 mM HEPES (pH 7.2), and 30 μl of 100 mM EDTA. The lowerorganic phase was dried under N₂. Ceramide-1 phosphate was resolved bythin-layer chromatography on silica gel 60 plates (Whatman) using asolvent system of chloroform:methanol:acetic acid (65:15:5, v/v/v) anddetected by autoradiography, and incorporated ³²P was quantified byliquid scintillation counting. The level of ceramide was determined bycomparison with a standard curve generated concomitantly of knownamounts of ceramide.

ASMase Activity Assay

ASMase activity was quantified by radioenzymatic assay using[¹⁴C-methylcholine]sphingomyelin (Amersham) as substrate as described(Schissel et al. J Biol Chem, 271, 18431-18436, 1996). Cell lysates wereincubated with [¹⁴C-methylcholine]-sphingomyelin substrate (0.026mCi/9.5 nmol) in 250 mM sodium acetate, pH 5.0 supplemented with 0.1%Triton X-100 and 1 mM EDTA or 0.1 mM ZnCl. Reactions were terminatedafter 1 h with CHCl3:MeOH:1N HCl (100:100:1, v/v/v), and[¹⁴C-methylcholine]phosphocholine product in the aqueous layer of theFolch extract was quantified using a Beckman Packard 2200 CA Tricarbscintillation counter. For detection of ASMase activity in patients, 10μl of serum was used.

CRM Detection by Confocal Microscopy

BAEC were grown on CC2-treated chamber slides (Nalge Nunc InternationalCorp., Naperville, USA) and then exposed to etoposide or paclitaxel withor without pre-incubation with Nystatin (30 μg/ml, Sigma) for 30 min.BAEC were then washed in cold PBS, fixed for 15 min in fresh 2%paraformaldehyde, washed 2× with cold PBS, and blocked with 5% FBS inPBS for 20 min at room temperature. Cells were stained withanti-ceramide MID 15B4 IgM (Alexis Corporation) at 1:50 dilution for 1hour at room temperature, washed 3× in PBS, and thereafter with TexasRed-conjugated secondary antibody (Jackson Immunoresearch Laboratories,Inc.) at 1:500 dilution for 1 hour at room temperature. Nonspecificfluorescence was excluded using isotype control IgM (BD Biosciences).Cells were washed 3×, stained with DAPI and mounted in 0.1%paraphenylenediamine. Fluorescence was detected using Leica TCS SP2 AOBS1- and 2-photon laser scanning confocal (DMRXA2 upright stand)microscope coupled with MetaMorph 7.5 (Molecular Devices). Number ofCRMs in membranes of endothelial cells was analyzed using MetaMorph 7.5software that allowed outlining of regions containing CRMs based on twocriteria: 1) CRM size (≥500 nm); 2) Intensity of ceramide staining.Mice: asmase^(−/+) and asmase^(−/−) mice, maintained in a SCIDbackground, were propagated using heterozygous breeding pairs andgenotyped as previously described (Santana et al. Cell, 86, 189-199,1996; Grassme et al. Nat Med 9, 322-330 (2003). 6-12 week-oldC57BL6/SV129 male mice, purchased from The Jackson Laboratory (BarHarbor).

Tumor Animal Models

Human HCT-116 colon cancer cells and MCA/129 murine sarcoma cells weremaintained in DMEM with 10% fetal bovine serum, with 100 u/ml penicillinand 100 μg/ml streptomycin. Cells were grown as monolayers in 75 cm²culture flasks at 37° C. in 10% CO₂ humidified incubators. Cells weretrypsinized, washed in PBS and diluted in Matrigel/PBS solution (40:60v/v) for HCT-116 xenografts or in PBS for MCA/129 syngeneic tumorgrafts. Cells (3×10⁶ for HCT-116 or 1×10⁶ for MCA129) were injectedsubcutaneously into the flanks of mice (Garcia-Barros et al. Science300, 1155-1159, 2003).

Tumor Response In Vivo

SCID^(asmase+/+) and SCID^(asmase−/−) mice harboring HCT-116 tumors(50-100 mm³) were treated with three i.p. injections of 35-50 mg/kgetoposide (Novaplus®), or 15-25 mg/kg paclitaxel (Hospira), or 1-6 mg/kgcisplatin (APP Pharmaceuticals, LLC) in a bi-weekly schedule with orwithout i.v. DC101 (1600 μg/25 gm mouse), or control IgG. For sarcomastudies, Sv129/B16 mice harboring MCA/129 tumors (100-150 mm³) weretreated with three i.p. injections of 240 mg/kg gemcitabine on abi-weekly schedule with or without i.v. DC-101. Tumor volume, based oncaliper measurements (length and width in millimeters), was calculateddaily using the following formula: 0.5×length×width².

Apoptosis In Vivo

To evaluate acute endothelial apoptosis, tumors were grown in mice andtreated with drugs as above. After the first treatment, tumors wereharvested at specified times, placed into formalin overnight andparaffin-embedded the next day. Paraffin-embedded sections (5 μm) wereco-stained for apoptotic endothelium using TUNEL and theendothelial-specific monoclonal antibody MECA-32 (Developmental StudiesHybridoma Bank, developed under the auspices of the NICHD and maintainedby The University of Iowa, IA).

Phase II Trial of Gemcitabine and Docetaxel with Bevacizumab in SelectedSarcoma Subtypes

Patients with metastatic or locally-recurrent leiomyosarcoma,pleomorphic undifferentiated sarcoma, pleomorphic liposarcoma,rhabdomyosarcoma or angiosarcoma, were treated on a phase II trial ofgemcitabine, docetaxel and bevacizumab. Initially the trial was adouble-blind, placebo-controlled, randomized trial of gemcitabine anddocetaxel with or without bevacizumab, which was modified to asingle-arm, open-label, non-randomized study of gemcitabine, docetaxeland bevacizumab. The protocol was approved by the Institutional ReviewBoard of Memorial-Sloan Kettering Cancer Center, and all patientsprovided written informed consent (Clinicaltrials.gov identifierNCT00887809).

Patients were treated with bevacizumab 15 mg/kg on day 1 of each 21-daycycle intravenously over 30 min with gemcitabine 900 mg/m² over 90 minon day 1 and 8 and docetaxel 75 mg/m² over 60 min on day 8. Initially,gemcitabine was administered immediately following bevacizumab. However,based on the results of our pre-clinical studies, the protocol was lateramended so that gemcitabine was administered at 1 hour afterbevacizumab.

Volumetric measurement of tumors was performed on an exploratory basisas the original study was not designed or powered to specificallyinvestigate the clinical application of timed anti-angiogenic therapy asa method of tumor chemosensitization. In light of the results from ouranimal studies, which were made part way through the clinical studyperiod, volumetric measurements were proposed to more ideally facilitatecomparison with our pre-clinical data. Furthermore, they likely providea more meaningful assessment of tumor response than the RECIST basedevaluation for soft tissue sarcoma. Therefore, on completion of thestudy, volumetric analysis was performed by a study radiologist (R.L.)blinded to assignment of patients to the bevacizumab administrationschedule or the initial placebo cohort. Baseline CT or MM was used toidentify a dominant target lesion for measurement. Follow-up scans wereevaluated for best response. Tumor contours were defined manually andsummed across all axial slices to calculate a total volume measurement.Tumor response was defined as at least a 30% reduction in tumor volume.Both a two-tailed Fisher's Exact Test and the Mann-Whitney Rank-Sum testwere employed to evaluate significance in patient volumetric responserates at the first interval treatment scan (week 6) and at time ofmaximum treatment response.

Microvascular Vasoconstriction as Ascertained for Example by PerfusionAlterations

Microvascular vasoconstriction was assessed by in situ tumor perfusionmeasurements using DCE-MRI with Gd-DTPA (Cho et al. Neoplasia 11,247-259, 2009). A syringe filled with the contrast agent Gd-DTPA (0.2 mMGd/kg, Magnevist; Berlex Laboratories, Inc., Wayne, N.J.) was connectedto the three-way stopcock through Gd-DTPA-filled tubing. The entireassembly including the anesthetized animal was positioned inside themagnet using a spirit level and the axial MR profile. Respiration wasmonitored during the MR experiment. The MRI coil was tuned and matchedto the proton frequency, followed by shimming of the sample. Tissueperfusion was calculated according to a model developed by Hoffmann etal. (Hoffmann et al. Society of Magnetic Resonance in Medicine 33,506-514, 1995). This model is based on the linear relationship betweenmeasured saturation recovery MR signal and the concentration of Gd-DTPAin the tissue. Akep value is analogous to the slope of time-dependent MRsignal enhancement and is considered an approximate measure of bloodflow/perfusion of the tumor tissue. Akep maps were generated each of thetumor slices. Additionally, quantification analysis of DCE-MRI studiesand their clinical applications are discussed by Padhani and Husband inDynamic contrast-enhanced MRI studies in oncology with an emphasis onquantification, validation and human studies (Clin Radiol. 2001 August;56(8): 607-20.)

Perfusion Measurements Using Hoechst 33342

The fluorescent dye Hoechst 33342 (5 mg/mL in physiologic saline; 15mg/kg; nominal injected volume, 0.1 mL; Sigma-Aldrich) was administeredvia tail vein injection at indicated times before and after treatment.

EPR Imaging

EPR imaging which measures oxygen levels in the tumor was carried out asdescribed previously (Epel et al. Concepts in magnetic resonance. PartB, Magnetic resonance engineering 33B, 163-176, 2008). The spin probeused for the EPR imaging was a 1 mM solution of OX063H radical(methyltris[8-carboxy-2,2,6,6-tetrakis[(2-hydroxyethyl]benzo[1,2-d:4,5-d′]bis[1,3]dithiol-4-yl]-trisodiumsalt, from GE Healthcare. The spin probe was enclosed in a borosilicateglass cylinder. Samples were deoxygenated by multiple-cyclefreeze-pump-thaw technique and flame-sealed. Samples were then placedinto the resonator, along the resonator's axis of symmetry and centeredin the axial plane of the resonator.

IVIM DW-MRI

IVIM DW-MRI was first described by Le Bihan et al. (MR imaging ofintravoxel incoherent motions: application to diffusion and perfusion inneurologic disorders. Radiology 1986; 161: 401-407). The novelty of ourapproach lies in repeatedly acquiring IVIM data over time (e.g.“dynamic” IVIM) to monitor the acute effects of radiation (orchemotherapy), a strategy which has not been previously reported. Nointravenous contrast administration is required, and IVIM parameters canbe measured many times, allowing us to define kinetics of vasculardysfunction after radiation or chemotherapy. Other alternatives, such asDCE-MRI or ¹⁵O-PET, would not serve our purpose as well: although theymay be highly reflective of tissue vascularity they cannot bere-injected into a patient to serially monitor changes in vascularityover time. IVIM parameters including perfusion fraction (f),pseudo-diffusion coefficient (D*) and diffusion coefficient (D) werecalculated for each lesion, using a biexponential signal decay model andincorporating a correction to account for differences in the T1 and T2relaxation times of tissue and blood, respectively.

In addition to DCE-MRI and IVIM DW-MRI, perfusion can be determinedusing any of the methods known in the art. ASMase or apoptotic ceramidelevels can be used as surrogates.

Ceramide Mass Spectrometry

Pro-apoptotic ceramide species (C16:0, C18:0) and anti-apoptotic species(C24:0) were measured for the studies included in this disclosure in theMSKCC Mass Spectrometry Core Facility according to standard procedures(see, e.g., Merrill, A. H. Jr., 2011, Chem. Rev. 111:6387-6642).

Example 1 Chemotherapeutic Agents Activate ASMase/Ceramide Pathway inEndothelial Cells

To test the effect of chemotherapy on ASMase signaling and ceramideproduction, BAEC were treated with paclitaxel (100 nM) and ASMaseactivity was determined by radioenzymatic assay (Rotolo et al, infra)using [¹⁴C-methylcholine] sphingomyelin (Amersham) as substrate (FIG.1A). Induction of ASMase activity was observed within 5 minutes aftertreatment, and the activation continued for 30 minutes. Ceramidegeneration was monitored using the diacylglycerol kinase assay(Stancevic et al. PLoS ONE 8, e69025, 2013). Consistent with what wasobserved with ASMase activity, ceramide generation peaked within minutesfollowing the treatment, and persisted for 30 minutes (FIG. 1B). Sinceceramide generation is associated with the formation of CRMs, BAECmonolayers were stained with anti-ceramide antibody (MID 15B4 IgM(Alexis Corporation) and CRM's were visualized using confocal microscopy(FIG. 1C). As anticipated, CRM formation peaked at 5 minutes, andremained elevated for 30 minutes (FIG. 1C). Nystatin is acholesterol-depleting agent which disrupts sphingomyelin-rich cellsurface raft microdomains, thus inhibiting ASMase targeting ofsphingomyelin, and CRM formation. Treatment of BAEC with both paclitaxeland nystatin prevented the formation of CRMs (FIG. 1C), showing thatnystatin's interference with ASMase substantially obliteratespaclitaxel-mediated ceramide generation and confirms thechemotherapeutic agent's proapoptotic action through ASMase signalingand ceramide production. In further support of this, pre-incubation ofBAEC with nystatin prior to treatment with chemotherapy inhibitschemotherapy-induced apoptosis (FIG. 3).

To determine the generality of the results observed, experiments werecarried out using an additional endothelial cell line, human coronaryartery endothelial cells (HCAEC); experiments were also performed usinga different chemotherapeutic agent. Treatment of BAEC with etoposide (50μM) resulted in activation of ASMase and ceramide formation within a fewminutes and the effects remained for 30 min (FIGS. 1D and 1E). Next,HCAEC were treated with paclitaxel (100 nM) (data not shown) andetoposide (50 μM) (FIG. 1F), and ceramide generation was evaluated.Consistent with the results noted when BAEC cells were treated withpaclitaxel and etoposide, treatment of HCAEC with either paclitaxel oretoposide leads to the generation of ceramide (FIG. 1F).

To further establish the generality of ASMase activation by variouschemotherapeutic agents, BAEC and HCAEC were treated with increasingdoses of cisplatin (0.1-50 μM) (data not shown). However, on contrary tothe other agents tested, cisplatin treatment failed to activate ASMaseand result in generation of ceramide. The cisplatin datanotwithstanding, taken together, these results confirm that variouschemotherapeutic agents having different mechanisms of action induceASMase activation and generation of ceramide. The ASMase/ceramidepathway was activated by chemotherapeutic agents that belong todifferent classes of chemotherapeutic agents, such as: taxanes,topoisomerase inhibitors, and nucleoside analog metabolic inhibitors.Since such chemotherapy agents are characterized by distinct mechanismsof action in tumor cells, it is surprising that a group of such distinctagents converges on a common molecular (ASMase/ceramide) axis inendothelium. The present confirmation that distinct chemotherapeuticagents activate the ASMase/ceramide pathway permits the presentinventors to take into account the results of experiments using singledose radiotherapy, which has been reported to activate the same pathway(Rao, S. S. et al., Axitinib sensitization of high Single DoseRadiotherapy. Radiotherapy and Oncology: Journal of the European Societyfor Therapeutic Radiology and Oncology 111, 88-93,doi:10.1016/j.radonc.2014.02.010 (2014)).

Example 2 ASMase Signaling is Required for Chemotherapy-InducedEndothelial Cell Apoptosis In Vivo

Since the ASMase/ceramide pathway is known to play a critical role inendothelial injury and dysfunction, BAEC and HCAEC were treated withpaclitaxel (100 nM), etoposide (50 μM), and cisplatin (0.1-50 μM), andendothelial apoptosis was assessed. Treatment of cells with paclitaxeland etoposide induced apoptosis within 2 hours of drug exposure (FIGS.2A and 2B). On contrary, cisplatin failed to trigger an appropriateapoptotic response in bovine endothelial cells in this experiment evenat high concentrations (50 μM) (FIG. 2C).

Taken together, these results suggest that ASMase is a determinantcomponent of chemotherapy-induced endothelial cell death.

Example 3 Pro-Angiogenic Factors Inhibit Chemotherapy-InducedEndothelial Cell Apoptosis

Various pro-angiogenic factors have been shown to inhibit ASMaseactivation in endothelial cells. To set the basis for experimentsinvolving evaluation of anti-angiogenic therapy in combination withchemotherapy, and the role of ASMase/ceramide pathway in this process,the effects of various factors on the chemotherapy induced cell deathwere assessed. BAEC were pre-incubated for 30 min with bFGF (2 ng/mL),VEGF (2 ng/mL) or nystatin (30 μg/mL) prior to treatment with etoposide(50 and apoptosis was evaluated after 8 hours using bis-benzamidetrihydrochloride staining (FIG. 3). While treatment with etoposide alonesignificantly induces apoptosis, as seen in the control sample,pre-incubation of BAEC with either basic fibroblast growth factor (bFGF;2 ng/ml), vascular endothelial growth factor (VEGF; 2 ng/mL), ornystatin (30 μg/ml) substantially inhibits etoposide-induced apoptosis(FIG. 3).

Example 4 ASMase Signaling is Required for Chemotherapy-InducedEndothelial Cell Apoptosis In Vivo

To assess the role of ASMase signaling in in vivo chemotherapy response,two mouse models were used, where two distinct cancer cell lines wereimplanted in wild type and ASMase deficient mice. First, HCT116 humancolorectal cancer xenografts were implanted into immunodeficientwild-type SCID^(asmase+/+) and ASMase deficient SCID^(asmase−/−) mice.Tumor-bearing mice were treated for 4 hours with a single dose ofpaclitaxel (25 mg/kg), and tumor sections were fixed and double-stainedfor endothelial surface marker MECA-32 (dark grey plasma membrane) andTUNEL (nuclear dark grey stain) (FIG. 4A). FIG. 4B shows thequantification of endothelial cell apoptosis. While tumors generated inSCID^(asmase+/+) animals exhibited robust endothelial apoptosis, tumorsxenografted in SCID^(asmase−/−) littermates were resistant tochemotherapy-induced endothelial cell apoptosis.

Next, a mouse model of MCA/129 fibrosarcomas was used to test theability of a different chemotherapeutic agent, gemcitabine, to induceapoptosis in ASMase deficient animals. Similar to results obtained usingthe colorectal cancer model, gemcitabine (240 mg/kg) inducedtime-dependent endothelial cell apoptosis in Sv129/BL6^(asmase+/+) butnot Sv129/BL6^(asmase−/−) mice (FIGS. 4C and 4D). Gemcitabine treatmentdid not cause tumor cell apoptosis, indicating that the effects arespecific to the endothelial cell compartment.

Collectively, these observations indicate that ASMase signaling isrequired for chemotherapy-induced endothelial cell apoptotic response invivo. Furthermore, it is observed that ASMase/ceramide pathway mediatestumor growth delay caused by chemotherapy agents that have the abilityto activate ASMase/ceramide signaling.

Example 5 ASMase Signaling is Required for Tumor Response toChemotherapy in Animal Models

Tumor response to chemotherapy in the colorectal HCT116 xenograft modelwas evaluated. SCID^(asmase+/+) or SCID^(asmase−/−) mice harboringHCT116 tumors (50-70 mm³) were treated with paclitaxel (15/20/25 mg/kgi.p.) three times biweekly. Consistent with the requirement for ASMasesignaling in endothelial cell apoptosis, activity of ASMase pathway wasrequired for the effect of chemotherapy on tumor growth delay. HCT116xenografts in SCID^(asmase+/+) mice treated with paclitaxel exhibitedcomplete tumor response after 10±1 days, while there was no significanttumor response in xenografts in SCID^(asmase−/−) littermates (FIG. 5A).To test that the ASMase requirement is mediated by the generation ofceramide, SCID^(asmase+/+) mice harboring tumors as in FIG. 5A weretreated with etoposide (35/35/50 mg/kg i.p.) biweekly in the presence ofanti-ceramide or isotype control antibody. As shown in FIG. 5B,intravenous injection of anti-ceramide IgM 1 hour before each etoposideinjection resulted in attenuation of tumor growth delay compared toanimals treated with isotype control antibody. These results indicatemandatory engagement of endothelial ASMase signaling in parenchymaltumor response to chemotherapeutic agents.

To further test the ability of cisplatin to affect endothelial cellapoptosis and tumor response in vivo, effects of cisplatin on tumorgrowth were evaluated. In this tumor model, high dose cisplatintreatment (3×6 mg/kg) did not induce significant HCT116 tumor growthdelay (FIG. 6F) nor tumor endothelial apoptosis (data not shown).

Example 6 Angiogenic In Vivo Tumor Chemosensitization is Dependent onTiming

Virions reports have demonstrated additive effects or synergy when AA Asare combined with chemotherapy. A previous study has shown thatanti-VEGFR2 IgG antibody DC101 temporarily de-represses endothelialASMase inhibited by VEGF, which is ubiquitously produced in tumors viahypoxia-mediated HIF-1α transcriptional activation of angiocrines(Truman et al. PLoS ONE 5, 2010). Thus, to further explore the notion ofendothelial ASMase signaling requirement in response to chemotherapy,DC101 was used in combination with paclitaxel treatment. HCT116 tumorsin SCID^(asmase+/+) mice, while slightly affected by 3×15 mg/kgpaclitaxel or 1600 μg DC101 alone, exhibited a synergistic tumorresponse when used together (40% complete responses) (FIG. 6A).Interestingly, anti-angiogenic synergism with chemotherapeutic agentsdepended on synchronized delivery of agents. No chemosensitization wasobserved following a single dose of 15 mg/kg paclitaxel if DC101 wasinjected 3-48 hours prior to paclitaxel (FIG. 6B, cisplatin data notshown). Likewise, there was no added benefit to that of chemotherapyalone if DC101 was injected at any time from 1-48 hours post paclitaxeltreatment (FIG. 6C). The synergism was observed only whenanti-angiogenic agent was injected 1-2 hours prior to chemotherapytreatment (this interval can be considered within the range 0.5 to 2hours post AAA administration).

Similar results were observed using MCA/129 murine fibrosarcoma tumorsimplanted in Sv129/Bl6^(asmase+/+) mice. DC101 (1.6 mg/25 gm mouse i.p.)was provided either 1 hour prior to or immediately preceding exposure togemcitabine (240 mg/kg i.p.). After 4 hours of treatment, tumors wereharvested and endothelial cell apoptosis quantified. DC101 wassuccessful in augmenting endothelial apoptosis and tumor growth responseonly when delivered 1 hour before, but not immediately preceding, eachgemcitabine dose (FIGS. 6D and 6E).

The results presented here indicate that chemosensitization occurredonly if anti-angiogenic agent was delivered 1-2 hours precedingchemotherapy, but at no other time preceding or after chemotherapy.Thus, there is a chemosensitization interval or window following theadministration of anti-angiogenic agent during which treatment withchemotherapy should occur.

Example 7 Synchronized Timing of Bevacizumab with Gemcitabine ImprovesTumor Response in Patients with Sarcoma

Results shown in Examples 1-6 revealed a significant role ofASMase/ceramide pathway in mediating and facilitating tumor response tochemotherapy. In order to elucidate a role of ASMase in clinical tumorchemotherapy, an Institutional Review Board-approved, prospective phaseII clinical trial of gemcitabine and docetaxel with the anti-angiogenicdrug bevacizumab in advanced soft tissue sarcoma was carried out atMemorial Sloan Kettering Cancer Center between June 2009 and April2012(MSKCC 09-015; Clinicaltrials.gov identifier NCT00887809). Patientsenrolled in the trial had metastatic or recurrent leiomyosarcoma,pleomorphic undifferentiated sarcoma, pleomorphic liposarcoma,rhabdomyosarcoma or angiosarcoma (FIG. 7A).

A total of 38 patients were treated. A subset of patients receivedplacebo instead of bevacizumab. All other patients were treated on day 1of each 21-day cycle with bevacizumab (15 mg/kg) delivered intravenouslyand gemcitabine (900 mg/m² over 90 min). On day 8, gemcitabine (900mg/m² over 90 min) was administered followed by docetaxel (75 mg/m2 over60 min), without bevacizumab. 16 patients were treated on the immediatebevacizumab-gemcitabine schedule (bevacizumab was deliveredintravenously over 30 min, followed by intravenous gemcitabine), 14 wereon the 1-hour interval schedule (bevacizumab was delivered 1 hour priorto gemcitabine), while 8 patients received placebo rather thanbevacizumab. Volumetric analysis of tumor response in the patients wasperformed by a study radiologist blinded to assignment of patients tothe bevacizumab/gemcitabine administration schedule. Baseline CT scan orMM was used to identify a dominant target lesion for volumetricmeasurement, followed by volumetric assessment on initial post-treatmentfollow-up scan (2 months) and over the treatment course (representativeCT time course of abdominal metastasis is shown in FIG. 7B showingregression of metastatic tumor).

While 38% of patients that received immediate consecutivebevacizumab-gemcitabine delivery exhibited significant tumor response(defined as >30% reduction in tumor volume), the response was notstatistically different from the 25% volumetric response observed in the8-patient placebo control group (FIG. 7C).

However, nearly all patients, 13 of 14 (93%), in thebevacizumab-gemcitabine 1-hour interval schedule cohort had asignificantly higher tumor response (FIG. 7D). Volumetric analysis wasfurther performed on the initial post-treatment follow-up scan (aftertwo 3-week treatment cycles). 4 of 16 patients or 25% of those receivingimmediate consecutive bevacizumab-gemcitabine delivery exhibitedsignificant tumor response (≥30% reduction in tumor volume), while 9 of14 (64%), in the bevacizumab-gemcitabine 1-hour interval schedule cohortdisplayed >30% tumor reduction.

These studies establish that synchronized timing of bevacizumab withgemcitabine, where bevacizumab is delivered 1 hour prior to theadministration of gemcitabine, significantly improves tumor response tochemotherapy in patients diagnosed with sarcoma. Overall, clinicaloutcome of soft tissue sarcoma patients in this trial representsproof-of-principle that ASMase signaling can be engaged for therapeuticbenefit. These results also support that ASMase signaling can beestablished as a biomarker for predicting tumor response and in anyevent for adjusting timing and dosage of the AAA, optimum timing of thechemotherapeutic drug (relative to the AAA administration) and dosage ofthe chemotherapeutic drug.

Example 8 Long-Acting Anti-Angiogenic Drugs Render Tumors Refractory toSubsequent Anti-Angiogenic ASMase-Mediated Tumor Sensitization

As patients undergo multiple rounds of long-acting anti-angiogenic drugtreatments in combination with chemotherapy, there is a progressiveincrease in the accumulated long-acting anti-angiogenic drug. Thus, theinventors tested whether the first round of long-acting anti-angiogenicdrug would impact the response of a second round, delivering full doseof long-acting anti-angiogenic drug in each cycle. Thus, the premise wasto evaluate whether a second round of attempted vascularchemosensitization at a time when bevacizumab levels would have decayedto half the level achieved after the first dose, exactly mimicking thefirst and second rounds of bevacizumab.

Briefly, 1×10⁶ MCA/129 fibrosarcoma cells were implanted into the rightflank of SW129/BI6^(JAx) mice and tumor volume was measured dailyaccording to the formula described by Kim et. al. (Cancer Res, 46,1120-1123 (1986). Mice were treated with gemcitabine (Gem) at 240 mg/kgi.p. twice at 4 day intervals (black arrows). Certain animals receivedDC101 (1600 mg/mouse=full dose) at 8 h and/or 1 h before each Gemtreatment, as indicated in FIG. 11A. Data (mean±SEM) were collated from5 mice/group. Another group of animals received either 0 or half of thedose of DC101 at 8 h hours prior to Gem treatment, and full dose ofDC101 at 1 hour prior to Gem treatment.

As shown in FIG. 11A, ASMase-based anti-angiogenic sensitization isattenuated when a full dose of a long-acting anti-angiogenic drug (suchas DC101) is already present in the subject at the time of attemptedASMase-based anti-angiogenic chemosensitization. On the contrary, whenonly half of the DC101 dose (800 mg/mouse) is present in the subject,the impact is minimal, and the inhibitory effect observed with the fulldose of DC101 is no longer detected. FIG. 11B shows the individual tumorresponse profiles as collated in FIG. 11A.

This study mimics the effects of accumulated long-acting anti-angiogenicagents such as bevacizumab, which occur during the successive rounds ofmulti-cycle clinical trial of a long-acting anti-angiogenic drugs incombination with chemotherapy. For example, at round two of a three-weekcycle, there would be half the dose of bevacizumab present in a subjectat the time of attempted ASMase-based chemosensitization. Furthermore,at round 4 of a three-week cycle, there would be even higher levels ofaccumulated long-acting anti-angiogenic drug, where the levels wouldcorrespond to approximately 88% of a full dose. At round 5, the levelsof accumulated long-acting anti-angiogenic drug would reachapproximately 94% of a full dose. These estimates are based on the factthat half-life (t_(1/2)) of an IgG, which bevacizumab is one of, is 4days in mice, and 21 days in humans.

This study provides evidence that long-acting anti-angiogenic agents(drugs) render tumors refractory to subsequent anti-angiogenicASMase-mediated tumor chemosensitization, where the strength ofinhibition intensifies as the amount of long-acting anti-angiogenicagent present in the subject increases. Thus, these experiments supportthe proposition that short-acting anti-angiogenic drugs with an averagehalf-life of about 120 hours or less are preferred for ASMase/ceramidepathway-based chemosensitization compared to long-actinganti-angiogenics such as bevacizumab or DC101.

Example 9 Kinetics and Dose Dependence of Gemcitabine-InducedASMase/Ceramide Pathway Activation

Pre-clinical models and clinical trial data presented in Examples 1-7showed that anti-angiogenic agents can substantially improve response ofdifferent tumors to different chemotherapy agents that activateASMase/ceramide pathway, when they are administered using a specificschedule that increases the activity of ASMase/ceramide pathway withinthe tumor microvasculature. In order to gain a more detailedunderstanding of principles that govern anti-angiogenicchemosensitization, studies testing the timing and dose dependence ofgemcitabine induction of sphingolipid signaling events in A19 BAEC willbe performed. 4 different readouts of ASMase/ceramide signaling in BAECwill be determined using standard protocols for these assays: 1)secretory ASMase activity, 2) ceramide generation, and 3) CRM formationwithin 60 min of gemcitabine administration, and 4) apoptosis at 2-24 h.Zn-dependent secretory ASMase activity will be measured by aradioenzymatic assay that uses [N-methyl-¹⁴C]sphingomyelin as substrate,under conditions that obey rules of Michaelis-Menten kinetics. When[¹⁴C]sphingomyelin is hydrolyzed, [¹⁴C]phosphocholine is released intothe aqueous phase of a Folch extraction, which is quantified byscintillation counting (Rotolo et al. J. Clin. Invest. 122: 1786-1790,2012). Cellular ceramide levels will be measured after Bligh and Dyerlipid extraction and analysis via liquid chromatography electrospraytandem mass spectrometry (Kasumov et al. Anal. Biochem. 401:154-161,2010). CRPs will be detected using confocal microscopy of fixed BAECcells incubated with anti-ceramide MID 15B4 IgM (Alexis Corporation)(Garzotto et al. Cancer Res. 58: 2260-2264, 1998).

Using a 90% lethal dose (LD₉₀) of gemcitabine, the time of maximumactivation for each readout will first be determined. Based on priorstudies, it is anticipated that ASMase/ceramide/CRP generation will peakwithin 5-15 min of gemcitabine administration and apoptosis will peak at6-8 hours. The experimentally determined peak times for each of the 4events will then be used to generate gemcitabine dose response curves,using a range of effective gemcitabine doses e.g., up to a 100 nM(Laquente et al. Mol. Cancer Ther. 2008; 7:638-647. Controls for ASMasespecificity will include pharmacologic ASMase inactivation usingimipramine (50 μM) and pre-treatment with the raft-disruptor nystatin(30 μg/ml). To delineate the order of early sphingolipid events,anti-ceramide 2A2 antibody, which does not block ASMaseactivation-ceramide generation, but prevents ceramide coalescence intoCRPs and apoptosis, will be used. In order to distinguish between ASMasesignaling and de novo ceramide synthesis, the well-defined ceramidesynthase inhibitor, Fumonisin B1, will be used (50 Collectively, theseexperiments will provide detailed information regarding the kinetics ofgemcitabine-induced sphingolipid death response in endothelial cells. Itis expected that ASMase activity will exhibit a statisticallysignificant increase in response to gemcitabine qualitatively similar tothat of FIG. 14C. Furthermore, ceramide levels (C16:0, C18:0) areexpected to undergo a statistically significant increase followingchemotherapy qualitatively similar to that of FIG. 8. This can be testedas described in Example 20.

Example 10 Determining the Timing of Anti-Angiogenic Administration andPeak of Gemcitabine Sensitization

Timing of DC101 and axitinib (selective inhibitor of VEGF receptors 1,2, and 3) chemosensitization of apoptosis will be optimized in culturedBAEC. Cells will be treated with DC101 (5 μg/ml) or axitinib (50 nM) ina time window encompassing −24 h to +24 h relative to the administrationof for example an LD₄₀ dose of gemcitabine. After that, the ability ofDC101 vs. axitinib to sensitize the dose-response curve for the fourreadouts will be assessed.

Example 11 Effect of Anti-Angiogenic Agents on a Subsequent Cycle ofChemotherapy

The clinical trial described in Example 7 above as well as preliminaryanimal data (not shown) prompted the hypothesis that prolongedanti-angiogenesis likely causes ASMase to become refractory to asubsequent round of anti-VEGF and chemotherapy treatment until decay ofthe anti-angiogenic effect re-sets ASMase sensitivity. For example, inthe Example 7 study, the majority of the benefit was achieved after onecycle of AAA+chemotherapy such that the second round of two-modalitytreatment was either of no additional benefit or minimal additionalbenefit. See FIGS. 7E and 7F. This hypothesis was confirmed in theexperiment of Example 8.

In order to further test the effects of anti-angiogenics on a subsequentcycle of chemotherapy, BAEC will be treated with maximal dose of thelong-acting AAA DC101 or the short-acting AAA axitinib, plus LD₄₀gemcitabine, according to the timing information obtained in Example 10.In order to remove endogenous anti-angiogenic agents, culture media willbe replaced at varying intervals (6 h to 2 days), after which cells willbe re-treated with gemcitabine±DC101 or axitinib. The effects ofsuccessive cycles of therapy on all four readouts described in Example 9will be evaluated. It is anticipated that these studies will determine aminimum time window necessary related to the decay time of the AAA andneeded to re-set ASMase sensitivity to anti-angiogenic de-repression.

Example 12 Effects of Long-Acting Vs. Short-Acting Anti-Angiogenic on a2nd Cycle of Chemotherapy

Since prolonged anti-angiogenic treatment renders ASMase refractory to asubsequent cycle of anti-VEGF treatment until decline in anti-angiogeniceffect re-sets ASMase sensitivity, it is possible that persistence ofcirculating long-acting anti-angiogenic from treatment cycle 1 willattenuate sphingolipid-based anti-angiogenic chemosensitization in cycle2. Thus, if this is the case, short-acting angiogenic drugs are likelyto be superior for ASMase/ceramide pathway based chemosensitizationcompared to long-lasting anti-angiogenics such as bevacizumab or DC101.

In vivo experiments using a mouse model of MCA/129 fibrosarcoma will beused to directly assess and compare the impact of both long andshort-acting anti-angiogenics on a subsequent round of chemotherapy.

For long-lasting anti-angiogenics, different intervals between thetreatment cycles will be tested, where the range evaluated will varyfrom 0.25-4 DC101 half-lives (1-16 days, half-life 4 days in mice). Itis important to note that half-life of IgG in mice is shorter than inhumans, 4 days vs. 21 days. Readouts of ASMase/ceramide pathway activitywill be determined, as well as the tumor growth response between the twocycles (assessed by standard caliper measurement as per Kim et al.(Cancer Res, 46, 1120-1123 (1986). In the initial set of experiments,animals will be treated with gemcitabine for 6 hours prior to theassessment of tumor endothelial apoptosis. Results from these intervaltiming studies of anti-angiogenic chemosensitization of endothelialapoptosis will be validated by examining full tumor response at 90 days,employing the same DC101 half-life regimen used for endothelialapoptosis, noting complete response and tumor growth delay.

Based on the findings presented in this disclosure, it is anticipatedthat gemcitabine-induced endothelial apoptosis is going to correlatewith tumor responses.

Example 13 ASMase Activity Predicts Tumor Response Outcomes FollowingHigh Single Doses of Radiation in the Murine MCA/29 Fibrosarcoma Model

Single dose radiotherapy (SDRT) has proven successful in treatingcancers previously considered radioresistant. While the exact mechanismof SDRT response has not been completely characterized, it is known thatthe endothelial cell ASMase/ceramide pathway plays a significant role inmediating this response. Radiation induces rapid endothelial cell ASMaseactivation, and radiation-induced apoptosis is ASMase-dependent, asmouse endothelium lacking ASMase is resistant to apoptosis. Furthermore,the defect in apoptosis is rescued by addition of exogenous ceramide.

In this Example, the inventors evaluated serum ASMase levels in aMCA/129 fibrosarcoma mouse model in commercial Sv129/BL6 mice followingirradiation. As shown in FIGS. 12A and 12B, serum ASMase activityincreases in a dose dependent manner following irradiation. Detailedcontrol dose studies determined the 50% tumor control dose (TCD₅₀) to be29.8 Gy+/−0.9 Gy as defined by complete response (no palpable tumor at30 days post-irradiation). As the time course experiment demonstratedelevations in ASMase at all time points from 1-72 hourspost-irradiation, the 24 hour time point was elected for furtherevaluation. Significant differences in serum ASMase activity elevationat 24 hours post-radiation were observed when 15, 27, and 40 Gy wereapplied, but no significant changes occurred after 9 Gy (FIG. 12C).Importantly, the SDRT-induced serum ASMase activity increased linearlyover the range of 15 to 40 Gy and correlated closely to completeresponse (R²=0.89, FIG. 12D), suggesting serum ASMase as a potentialbiomarker of ASMase/ceramide pathway activity in clinical SDRTscenarios.

In the second part of the study, the inventors tested a hypothesis thatASMase/ceramide pathway can be used as a biomarker in SDRT response.Using a uniform dose of 27 Gy, slightly below the TCD₅₀ of the MCA/129model in Sv129/BL6 mice (in an extended cohort of Sv129/BL6 mouse mice(n=19)) the inventors evaluated whether induced serum ASMase activity at24 hours predicted tumor response at 30 days. In these studies, fivemice exhibited complete response, while three additional mice exhibitedpalpable tumors that did not grow for >15 days following radiation,defined as partial response. The remainder of the cohort continued togrow and were defined as no response. Every mouse exhibited an increasein serum ASMase activity post irradiation, ranging from a 1.1 to 2.7fold increase (FIG. 13A). However, for tumors that exhibited a completeor partial response to SDRT (light gray and dark grey bars, FIG. 13A),higher fold changes in ASMase activity were observed (FIGS. 13A, 13B),directly linking response to ASMase/ceramide pathway activation.

As shown here, the increase in ASMase serum activity was proportional toSDRT response. This is important as it indicates that targeting factorsknown to inhibit ASMase, such as angiogenic growth factors (for exampleVEGF signaling) would lead to further increase in ASMase, which in turnwould result in an enhanced response to SDRT. Thus, use ofanti-angiogenic agents (shortly before SDRT treatment) capable offurther increasing and/or activating ASMase during the SDRT treatment ishighly desirable. In order to achieve even greater ASMase activation, itwould be attractive to use a higher dosage of anti-angiogenic agentwhich has also been shown here to increase ASMase activity during ashort-duration window (especially since the other therapeutic modality,SDRT or chemotherapy, is often administered at or near the maximumtolerated dose). For example, the maximal ASMase increasing dose of theAAA (subject to any dose-limiting toxicity) can be assessed andadministered. It is anticipated that this will be substantially higherthan the low daily dose formulations currently available.

Accordingly, increase of ASMase activity as much as feasible withoutinducing toxicity in the treated subject emerges as a goal of treatmentoptimization, by dosage adjustment of ASMase stimulating agents, whetherit be the AAA or the SDRT or the chemotherapy modality or both.

It emerges from the foregoing data that activation of the ASMase pathwaywhich leads to ischemia reperfusion injury and permits tumor regressionand subject survival is common to both HDRT and chemotherapy. Theability of AAA to attenuate ASMase and therefore attenuate the intensityof ischemia reperfusion, except if given at the appropriate timerelative to the second modality, is also established. Accordingly,results described herein regarding ASMase activation and ischemiareperfusion intensity are applicable to either combination: AAA+SDRT orAAA+chemotherapy

Example 14 Single Dose Radiotherapy Induces MicrovascularVasoconstriction in Animal Models

Recent studies have shown that anti-angiogenic drugs can de-repressASMase/ceramide pathway to enhance ceramide-mediated endothelialapoptosis, but only if delivered prior but close to the time of SDRT,e.g., an hour and in any event less than two hours before SDRT (Rao etal. Radiotherapy and oncology: journal of the European Society forTherapeutic Radiology and Oncology 111, 88-93,2014). In animal models,xenograft responses to SDRT combined with anti-angiogenic treatment aresignificantly attenuated when tumors are implanted in asm^(−/−) mice.Similar effects are observed when asm^(+/+) mice bearing xenografts arepre-treated with an inhibitory anti-ceramide antibody. These resultsindicate that host endothelium is an important component of SDRT-inducedtumor responses combined with direct effects of radiation on tumorcells.

Single dose radiotherapy (SDRT) has proven successful in treatingcancers previously considered radioresistant. While the exact mechanismof SDRT response has not been completely characterized, it is known thatthe endothelial cell ASMase/ceramide pathway plays a significant role inmediating this response. Radiation induces rapid endothelial cell ASMaseactivation, and radiation-induced apoptosis is ASMase dependent, asendothelium of mice lacking ASMase is resistant to apoptosis.Furthermore, the defect in apoptosis is rescued by addition of exogenousceramide.

Recent studies have shown that anti-angiogenic drugs can de-repressASMase/ceramide pathway to enhance ceramide-mediated endothelialapoptosis, but only if delivered prior but close to the time of SDRT,e.g., an hour and in any event less than two hours before SDRT (Rao etal. Radiotherapy and oncology: journal of the European Society forTherapeutic Radiology and Oncology 111, 88-93, 2014). In animal models,xenograft responses to SDRT combined with anti-angiogenic treatment aresignificantly attenuated when tumors are implanted in ASMase^(−/−) mice.Similar effects are observed when asmase^(+/+) mice bearing xenograftswere pre-treated with an inhibitory anti-ceramide antibody (data notshown). These results indicate that host endothelium is an importantcomponent of SDRT-induced tumor responses combined with direct effectsof radiation on tumor cells. To gain a better understanding of thebiological processes that govern vascular dysfunction caused by SDRT, itwas postulated that ASMase/ceramide pathway mediates microvascularvasoconstriction as an acute injury response on a pathway to apoptoticdeath. Thus, tumor microvascular vasoconstriction (measured, e.g., byperfusion alterations) was evaluated following different doses of SDRT.

SDRT induced vascular dysfunction was assessed by in situ tumorperfusion measurements using DCE-MRI with Gd-DTPA (Cho et al. Neoplasia11, 247-259, 2009). Tissue perfusion was calculated according to a modeldeveloped by Hoffmann et al. (Hoffmann et al. Society of MagneticResonance in Medicine 33, 506-514, 1995). Akep values of the whole tumorsections were determined, wherein a decrease in Akep values wasindicative of perfusion reduction and hence tumor hypoxia or ischemia. Ashort time (30 min) after radiation, tumor samples (two different tumortypes were tested) from the asm^(+/+) host displayed significantperfusion reduction, while no reduction was detected in the sample fromthe asm−/− host. Similar perfusion dysfunction was not detected in theasm−/− host. Timing of ASMase-regulated vasoconstriction was quantifiedin the MCa allogeneic breast cancer model, wherein perfusion wasdecreased at 30 minutes and 100 minutes post-SDRT. The results wereconfirmed by measuring tumor capillary perfusion which significantlydecreased within a short time (30 min) after SDRT in asm^(+/+) micewhile it was not changed in asm^(−/−) mice. (Data not shown.) Electronparamagnetic resonance (EPR) spectroscopic 02 levels, showed 29%reduction in B16 melanoma O₂ tension post 20 Gy SDRT of tumors inasmase^(+/+) mice, abrogated in asmase−/− littermates.

Collectively, these results suggest acute perfusion deficits accompaniedby 02 decrements after SDRT. These observations put forward a plausiblemechanism by which ASMase-mediated endothelial dysfunction determinestumor response, in which apoptosis per se does not account for tumorresponse; rather ASMase-mediated acute vascular dysfunctiondistinguishes SDRT from conventional fractionated radiotherapy. Theseobservations can be extrapolated to chemotherapy as ASMase is alsoincreased in response to chemotherapy.

Example 15 Microvascular Vasoconstriction Occurs within 1-2 HoursFollowing 24 Gy Single Dose Radiotherapy

Vascular dysfunction in patients with metastatic disease was assessedusing a non-invasive imaging technique known as intravoxel incoherentmotion (IVIM) diffusion-weighted magnetic resonance imaging (IVIMDW-MRI). In biologic tissues, microscopic motion detected by standardDW-MRI includes (i) diffusion of water molecules, influenced bystructural components of tissue, and (ii) microcirculation of blood inthe capillary network (perfusion). In tissues characterized by highcellular density, such as tumors, motion of water molecules is moreconstrained than in normal tissues.

IVIM-DW-MRI allows for acquisition of multiple measurements over a shorttime period. In IVIM DW-MRI, multiple b-values are applied, reflectingdifferent strengths and timing of the diffusion gradient. Therelationship between the MR signal intensity (S) within a tumor atdifferent b values exhibits a bi-exponential pattern with a steep slopeat low b-values and a shallower slope at higher b-values. The steepnessof the initial curve at low b-values reflects the effect ofmicrovascular events on diffusion. The initial slope of the curverepresents an estimate of flow velocity (pseudodiffusion, D*) within themicrovasculature and 1 minus the Y-intercept of the later portion of thecurve represents the perfusion fraction (f), i.e. the volume of bloodwithin the microvasculature. Thus, both parameters provide relatedmeasures of microvascular function. In 15 patients (10 of whom alsoelected to participate in the serum collection), IVIM DW-MRI wasperformed before radiation and repeated within 0.5-2 hours followingradiation, a time frame selected based on the pre-clinical data thatdemonstrates ASMase-dependent reduction in perfusion at 0.5-2 hoursfollowing SDRT. The IVIM image was acquired at several consecutive timepoints with 4 min intervals between acquisitions. An experiencedradiologist placed a volume of interest (VOI) on the irradiated lesionon the IVIM DW-MRI images, using anatomic (T1- and T2-weighted) MRIsequences as a reference (FIG. 9A). D* and f values were calculatedwithin each pixel of the VOI and averaged to produce a mean tumor D* andf. Up to 16 repeated mean tumor D* and f measurements were obtained pre-and post-SDRT. The pre-radiation IVIM-DW MRI D* and f values exhibitedconsiderable heterogeneity between tumors, reflecting differing baselinetumor microvascular perfusion (FIGS. 9B and 9C). To compare changes inmicrovascular perfusion, mean tumor D* and f are expressed as a fractionof the each tumor's pre-SDRT mean tumor D* and f (FIGS. 9D, 9E). In the9 patients receiving 24 Gy SDRT, there was significant decrease in IVIMparameters f (mean−30%) and D* (mean−24%). In contrast, in the 6patients that underwent IVIM imaging before and after 9 Gy, mean D* andf were not substantially changed. These results suggest MRI detectableperfusion deficits may serve to biomark the tumor biology of SDRT.

These findings could be expanded to include more doses, which would thenallow for the establishment of minimum and maximum doses of SDRT thatinduce tumor microvascular vasoconstriction.

Example 16 Increased ASMase Activity is Detected in Serum of Patients1-2 Hours Following 24 Gy SDRT

Recent studies have shown that anti-angiogenic drugs can de-repressASMase/ceramide pathway to enhance ceramide-mediated endothelialapoptosis when delivered 1-2 hours before SDRT (Rao et al. Radiotherapyand oncology: journal of the European Society for Therapeutic Radiologyand Oncology 111, 88-93, 2014). As the data presented here indicate thatSDRT leads to microvascular vasoconstriction after 24 Gy, ASMase wasmeasured following SDRT.

Several dose and fractionation schemes are used in clinical treatment ofbone metastases to the spine. Common regimens include single doses of16-24 Gy and hypofractionated regimens such as 9 Gy×3 fractions. It isnot known whether one regimen is superior to another in terms of localtumor control. This question is the focus of an ongoingmulti-institutional randomized trial (NCT01223248) comparing 24 Gy×1fraction to 9 Gy×3 fractions for patients with bone metastases of anysolid tumor histology with the primary endpoint of local control. In asubset of patients from this trial who consented to a nested biomarkerstudy, serum samples were collected before, and at 1 and 24 hoursfollowing radiation treatment. For the 9 Gy×3 arm, serum was collectedafter the first 9 Gy radiation dose. This strategy allowed for directtesting of the hypothesis, developed pre-clinically, that there is adose threshold for activation of the ASMase/ceramide pathway. In the 18patients accrued to the biomarker substudy, 10 were accrued within the24 Gy cohort and 8 within the 9 Gy×3 cohort (FIG. 10C). As hypothesized,there was an elevation in serum ASMase activity following 24 Gy, but nochange following 9 Gy (FIGS. 10A, 10B). Seven of 10 patients in the 24Gy group exhibited increases in ASMase activity (1.2 to 1.5 foldincreases, FIG. 10A). Elevations were most prominent and consistent atthe 24 hour time point (FIG. 10B), but nonetheless mean values wereelevated at both 1 and 24 hours post-SDRT, consistent with ourobservations in the MCa/129 murine fibrosarcoma model.

To examine whether an induced serum ASMase activity increase might occurafter the total cumulative radiation dose reached a specific thresholdin the range of 24 Gy, we examined ASMase activity levels in 5 patientsamples at 1 and 24 hours following the 3^(rd) fraction of 9 Gy(cumulative dose of 27 Gy). No significant ASMase serum changes wereseen above baseline at 1 hour (mean 1.04±0.06 fold change) or at 24hours (mean 1.02±0.07 fold change) after the third dose of 9 Gy. Thesestudies provide strong support for the hypothesis that rapid release ofASMase into the circulation can biomark the biologic impact of SDRT inpatients.

Thus, measured ASMase activity may constitute an alternative to IVIMDW-MRI as a biomarker of SDRT clinical response.

Example 17 Microvascular Vasoconstriction Occurs Immediately FollowingAdministration of a Chemotherapeutic Agent Activating ASMase Signalingin Animal Model

Based on the results described in Example 14, wherein the activation ofthe ASMase/ceramide pathway by radiotherapy mediates vasoconstrictionand given the results of Example 14, it was postulated thatvasoconstriction and ensuing vascular perfusion defects also occurfollowing administration of chemotherapeutic agents that activate thesame pathway. In order to confirm this, vascular dysfunction wasassessed by measuring perfusion/permeability as described in Example 14.

Briefly, MCA/129 fibrosarcoma tumors were implanted in the rear mousehip, and subjected to intravoxel incoherent motion (IVIM) MRI whentumors reached a size of 150-300 mm³. After i.p. injection ofgemcitabine (240 mg/kg) both vascularity (Fp, FIG. 14A) and diffusion(D*, FIG. 14B) were reduced in a reversible manner over a 1 hour timeperiod, where the peak of vascularity and diffusion reduction wasobserved between 15-30 minutes following the treatment with gemcitabine(p<0.05, Bonferroni correction applied). Data (mean±SEM) are collatedfrom 6 animals per group.

Gemcitabine was able to activate ASMase in cultured endothelial cells.The experiment was conducted in order to study the time-course of ASMaseactivity after addition of 100 nM gemcitabine to bovine aorticendothelial cells. As shown in FIG. 14C, gemcitabine triggered rapidcellular activation of ASMase. Furthermore, the activation of ASMaseoccurred in a dose-dependent manner as evident in FIG. 14D, whichrepresents a dose-response curve of ASMase activation at 5 minutes.ASMase activity was determined on total cell lysates. Data (mean±SEM)are collated from 3 separate experiments.

The fact that ASMase activation occurs in a dose-dependent manner asshown in FIG. 14D is of importance, as it suggests that higher doses ofchemotherapy drugs might be needed in order to take full advantage ofASMase-mediated chemosensitization. Further studies are needed in orderto determine the optimal dose required and feasible.

Example 18 Establishing Vasoconstriction Immediately FollowingAdministration of a Chemotherapeutic Agent Activating ASMase Signalingin Asmase^(−/−) Animals

Given the findings that microvascular vasoconstriction occursimmediately following administration of a chemotherapeutic agent (FIGS.14A and 14B) the following study will confirm the role of ASMasesignaling in an additional animal model using asmase^(−/−) animals.

MCA/129 fibrosarcoma and B16 melanoma tumor models will be generated inasmase^(+/+) or asmase^(−/−) animals, followed by the treatment of micewith X (e.g., 1, 2, 3) doses of various chemotherapeutic agents thatactivate ASMase/ceramide signaling, such as paclitaxel, etoposide,and/or gemcitabine. Blood perfusion/permeability in tumor tissue will bedetermined at time intervals immediately prior to and within 0.5-24hours following injection of a bolus of chemotherapeutic agent.Perfusion reduction following the administration of chemotherapeuticagent in tumors implanted in asmase^(+/+) but not asmase^(−/−) animalswill confirm that activation of the ASMase-ceramide pathway mediatesmicrovascular vasoconstriction-dysfunction and therefore interferes withthe ability of the tumor to recover from the direct cytotoxic effects ofthe therapeutic agent.

In addition to MM detection, chemotherapy-induced perfusion defects willbe characterized using Hoecht Dye Extravasation (Chaplin et al. CancerResearch, 47, 597-601, 1987). Briefly, Hoecht 33342 will be injected viatail vein into asmase^(+/+) or asmas^(−/−) mice bearing MCA/129fibrosarcomas and B16 melanomas following the treatment withchemotherapy. Mean fluorescence of tumor sections will be used as ameasurement of tumor capillary perfusion. Measurements will be taken atvarious time-points post chemotherapy treatment.

Vascular perfusion will be determined using a third technology, whereelectron paramagnetic resonance (EPR) spectroscopic O₂ levels will bequantified by direct imaging of tumors in vivo (Epel et a. Concepts inmagnetic resonance. Part B, Magnetic resonance engineering 33B, 163-176,2008).

Collectively, these studies will provide detailed information regardingthe timing and dosage of chemotherapy and correspondingvasoconstriction, as well as the involvement of the ASMase-ceramidepathway in this process. It is anticipated that these experiments willconfirm that this pathway (through direct or indirect measurement (orderivation) of ASMase, sphingolipid, or ceramide) will provide importantbiomarkers of chemotherapy effectiveness.

Example 19 Testing Whether IVIM Diffusion-Weighted MRI canQuantitatively Assess Tumor Vascular Dysfunction Immediately afterChemotherapy and Serve as a Biomarker to Predict Tumor Response

Clinical trial data presented in Example 7 underscore the importance oftiming the administration of an anti-angiogenic agent with that of achemotherapeutic agent that activates the ASMase-ceramide pathway inorder to improve tumor response in patients. As disclosed herein,anti-angiogenic agent administration should be timed as to result inASMase-ceramide pathway activation. It is hypothesized that in additionto mediating endothelial cell apoptosis, ASMase-ceramide-signaling maygovern acute vascular dysfunction leading to rapid reduction in tumorperfusion followed by re-perfusion. This hypothesis was initially testedin animal models as described above in Example 17 and Example 18. Next,the occurrence of vascular dysfunction following timed anti-angiogenicand chemotherapy treatment in patients will be investigated.

Vascular dysfunction will be assessed using dynamic IVIMdiffusion-weighted magnetic resonance imaging (IVIM DW-MRI). In biologictissues, microscopic motion detected by standard DW-MRI includes (i)diffusion of water molecules, influenced by structural components oftissue, and (ii) microcirculation of blood in the capillary network(perfusion). In tissues characterized by high cellular density, such astumors, motion of water molecules is more constrained than in normaltissues. One of the important advantages of IVIM DW-MRI is that itallows repeated “dynamic” measurement of perfusion and diffusion-relatedsurrogate metrics every few minutes, without the need for intravenouscontrast agent. While standard DW-MRI is typically performed using asingle-shot spin-echo echo planar imaging (SE-EPI), a modified methodwill be used in these studies. A technique will be adapted as to allowfor (i) acquisition of multiple contiguous 2D slices, (ii) multipleb-values, and (iii) data censorship and off-line averaging. Since IVIMparameters can be measured many times over a short period of time (45-60minutes), IVIM DW-MRI can be used to obtained detailed kinetics of tumorvascular dysfunction following the administration of chemotherapy. Whileother alternatives, such as dynamic contrast-enhanced (DCE)-MRI or⁵¹O-PET could also be used to assess tissue vascularity, these methodsare not suitable for the purposes of the proposed study, as they cannotbe re-injected into a patient to serially monitor changes in vascularityover time.

Patients treated for metastatic disease to bone or soft tissue at MSKCCwill be recruited for the study. The following inclusion criteria willbe used: (i) histologically proven metastatic disease; (ii) patientsdeemed clinically appropriate for chemotherapy treatment; (iii) lifeexpectancy >6 months; (iv) age >18 years. Exclusion criteria: (i) unableto give informed consent; (ii) unable to comply with the protocol, (iii)MRI is contraindicated; (iv) tumors involving visceral organs, brain orspinal cord; (v) platelet count <75,000/μl, HgB level <9 g/dl, WBC<3500/μl; (vi) metastases in the upper thoracic spine (to avoid MMartifacts due to cardiac motion); (vii) lesions <1.5 cm (to ensurerobust measurements). Currently, patients with bone and soft tissuemetastases receive intravenous gemcitabine over a 90 minute period.

IVIM DW-MRI will be repeated 16 times following the intravenousadministration of 900 mg/m² of gemcitabine, or 75 mg/m² docetaxel whereimages will be acquired at various time points, including 60, 90, 120,and 150 minutes following chemotherapy treatment.

Perfusion fraction, pseudo-diffusion coefficient, and diffusioncoefficient will be calculated for each lesion, using a biexponentialsignal decay model and incorporating a correction to account fordifferences in the T1 and T2 relaxation times of tissue and blood,respectively (Lemke et al. Magnetic resonance in medicine: officialjournal of the Society of Magnetic Resonance in Medicine, 64, 1580-1585,2010). For patients in each drug (gemcitabine or docetaxel) category,post-treatment measurements (expressed as fractions of the pre-treatmentvalues) will be summarized and the nadir value/time point will bedetermined using time series plots. To test whether measurements at anyparticular time point are significantly lower than 1, one-sample,one-sided t-tests will be used. To test whether measurements at one timepoint are significantly lower than those at another time point(applicable for statistically confirming the nadir point), one-sidedpair-wise t-tests will be used.

The resulting detailed understanding of the kinetics ofchemotherapy-induced vascular injury will be used to develop an imagingbiomarker that will be used to optimize the doses and timing ofanti-angiogenics and chemotherapy. It is expected that IVIM parameters fand D*, which represent blood volume fraction and microcirculatoryperfusion of blood within capillaries, respectively, will be changedfollowing chemotherapy. The changed values are expected to fall into thefollowing ranges: 1) 30-50% reduction in blood volume fraction (f) (forexample, 40%); and 2) 15-35% reduction in microcirculatory perfusion ofblood within capillaries (D*) (for example, 25%).

Example 20 Testing Whether ASMase Activity and Quantity of CeramideSpecies Immediately Following Chemotherapy can Serve as a Biomarker andPredict Tumor Response to Chemotherapy

Findings disclosed herein suggest that activation of ASMase signaling aswell as generation of pro-apoptotic C16:0 and C18:0 ceramide may serveas biomarkers that can be used for chemotherapy schedule and doseoptimization, in combination with ASMase/ceramide sensitizinganti-angiogenic agents. The secretory form of ASMase, released inresponse to various chemotherapy agents, can be detected in human serum.In addition to ASMase, C16:0 or C18:0 ceramide can also be measuredusing human serum by mass spectroscopy (MS).

The inventors have already conducted animal studies regarding the serumceramide levels following irradiation. MCA129 fibrosarcoma tumorallografts (tumor size ˜150 mm3) were treated with 27 Gy IR. Mice werebled 24 h pre-irradiation (Oh) and 24 h post-irradiation (24 h) andserum harvested for MS analysis. As shown in FIG. 8, both C16:0 andC18:0 ceramide species were elevated in response to irradiation. On thecontrary, serum levels of anti-apoptotic ceramide C24:0 remainedunchanged following irradiation.

Given the similarities between ASMase activity increase in response toirradiation and ASMase activity increase in response to chemotherapy, itis anticipated that both pro-apoptotic C16:0 and C18:0 ceramide levelswill be increased in serum of animals and/or patients followingtreatment with chemotherapy agents.

In order to confirm that ASMase/ceramide pathway can serve as abiomarker in a clinical setting, patients undergoing IVIM DW-MRI studiesdescribed in Example 19 will have serum samples collected 1 hour priorand 24 hours after chemotherapy treatment.

8-10 ml of whole blood from each patient enrolled in the study will becollected into glass, anti-coagulant-free tubes and allowed to clot20-30 minutes. Following centrifugation of the sample at 1200 g, theserum supernatant will be stored under N₂ gas in 5×5000_, and 5×50 μLaliquots. ASMase activity will be assessed using 10 μL of serum withC14-labeled sphingomyelin as substrate. ASMase-mediated sphingomyelinhydrolysis leads to release of C14-labeled phosphocholine, which can beextracted into an aqueous phase and quantified by scintillationcounting. Furthermore, ceramide MS will be performed to assess bothpro-apoptotic (C16:0, C18:0) and anti-apoptotic ceramide species(C24:0).

It is anticipated that chemotherapy agents will cause a statisticallysignificant increase in ASMase levels and/or activity. Similarly,proapoptotic ceramide levels (C16:0, C18:0) are expected to show astatistically significant increase following chemotherapy.

Example 21 Clinical Trial for Dose Escalation of a Short-ActingAnti-Angiogenic Agent for Optimum Enhanced Chemosensitivity

A phase IB will be pursued in standard 3+3 format. As such, the first 3patients accrued to the trial will be treated at dose level 1 asprovided in the table below. Patients will be monitored fordose-limiting toxicity as well as for ASMase activation, acute effect ofanti-angiogenic agent on tumor vascularity measured for example by IVIMDW-MRI and of course tumor response measured for example by conventionalMill with contrast agent. ASMase levels will be measured by standardizedradioenzymatic assay using [14C]sphingomyelin as substrate as per Rao etal. (Radiotherapy and oncology: journal of the European Society forTherapeutic Radiology and Oncology 111, 88-93, 2014) or alternatively bymeasuring ceramide species, as described for example in Merrill, A. H.,Jr. (2011). Chem. Rev. 111, 6387-642. This clinical study will begenerally based on Rugo, H. S. et al, J. Clin. Oncol, 23(24): Aug. 20,2005.

If none of these patients experience a dose-limiting toxicity (DLT)after the first three cycles of treatment, the dose of AAA will beraised to dose level 2. If 2 out of 3 patients experiences DLT, the doselevel will be reduced and 3 new patients will be accrued at dose level−1. If 1 out of 3 patients experiences toxicity on dose level 1, afurther 3 patients will be accrued to this level. If <2/6 patients ondose level 1 experiences dose-limiting toxicity (DLT) the dose will beraised to dose level 2, as described below. If 2/6 patients on doselevel 1 experiences DLT, the dose level will be reduced and 3 patientswill be accrued at dose level −1. If 0/3 patients on dose level −1 thenexperiences DLT, this level would then be considered MTD. If 1/3patients experiences DLT, the dose level will be expanded to a total of6 patients. If <3/6 patients have DLT at this level, it will beconsidered the MTD. If >2 patients have DLT at dose level −1, thetreatment combination will be considered unfeasible and furtherdevelopment of this combination will be stopped. Conversely, if at doselevel 2 there is no DLT in the first 3 patients (or <2 out of 6patients), the dose will be increased to dose level 3. If toxicity isseen in >2 of the first 6 patients treated at this dose 2, dose level 1would be considered the MTD. If 0/3 or <2/6 patients treated at doselevel 2 has DLT, this will be considered the MTD. At the same time,ASMase increase will be monitored. Doses will continue to be escalateduntil the maximal ASMase activity level is reached immediately followingAAA administration, provided of course that DLT has not been reached.

The purpose of the ASMase measurements will be to assess whether anyadditional anti-angiogenic agent will result in an incremental increasein ASMase levels (or an incremental increase in ceramide specieslevels). If no additional ASMase is expressed with a higher AAA dose,there is no reason to increase the AAA dose even if DLT has not beenreached. If DLT is reached at a dose lower than the maximum ASMaseincrease dose, the dose dictated by DLT may be expected to be adopted.

ASMase measurements will continue to be taken to assess response to theAAA, i.e., to ensure that the administration of the chemotherapeuticagent will be to confirm that chemotherapy in each cycle will beadministered during the de-repressed ASMase interval or window and toconfirm that the AAA from the prior cycle will have decayed beforeadministration of another dose of AAA (and chemotherapy).

Each of the accrued patients will have serum samples collected 1 hourbefore and 24 hours after the first chemotherapy treatment. For eachpatient, 8-10 ml of whole blood will be collected into glass,anti-coagulant-free tubes and allowed to clot 20-30 minutes. Blood willbe centrifuged at 1200 g and the serum supernatant stored under N2 gasin 5×500 uL and 5×50 uL aliquots. ASMase activity assays are performedwith 10 μL of serum with C14-labeled sphingomyelin as substrate. ASMasehydrolyzes sphingomyelin, releasing C14-labeled phosphocholine that canbe extracted into an aqueous phase and quantified by scintillationcounting. Ceramide mass spectrometry (MS) will be performed usingalready established protocols (Merrill, A. H., Jr. (2011). Chem. Rev.111, 6387-6422) to assess pro-apoptotic (C16:0, C18:0) andanti-apoptotic ceramide species (C24:0).

Similarly the purpose of IVIM DW-MRI which allows repeated “dynamic”measurement of perfusion and diffusion-related surrogate metrics everyfew minutes, without the need for intravenous contrast is to assesstumor vascular dysfunction immediately upon administration of the AAAand chemotherapy. Based on preliminary data from SDRT, vascular volumefraction and vascular flow are reduced following treatment with combinedAAA and SDRT. It is anticipated that similar results will obtainfollowing the timed administration of AAA followed by chemotherapy inaccordance with the present disclosure. See for example Bisdas, S. etal. Correlative assessment of tumor microcirculation usingcontrast-enhanced perfusion MRI and intravoxel incoherent motiondiffusion-weighted MRI: is there a link between them? NMR in Biomedicine27, 1184-1191, doi:10.1002/nbm.3172 (2014).

Microcirculatory perfusion of blood within capillaries has no specificorientation and can be thought of as “pseudo-diffusion,” which dependson the velocity of the flowing blood and the vascular architecture. TheIVIM approach assumes that the measured MR signal attenuation comprisesa mixture of tissue perfusion and tissue diffusivity. These effects arecharacterized by using a bi-exponential function to model the MR signaldecay as a function of b-value instead of a mono-exponential decay.Acquiring IVIM images over an entire tumor takes 2-3 minutes and allowscalculation of quantitative indexes which describe tissue waterdiffusivity (D), tissue perfusion (pseudo-diffusion coefficient—D*), andvascular volume fraction (f). The ability of IVIM to non-invasivelyquantify “perfusion” (IVIM parameters D* and f) is central to theconcept of detecting of acute chemotherapy-induced (or combined AAA- andchemotherapy-induced) vascular dysfunction.

IVIM DW-MRI will be obtained at baseline and at various time points toassess response to the AAA, i.e., to ensure that the administration ofthe chemotherapeutic agent will be to confirm that chemotherapy in eachcycle will be administered during the de-repressed ASMase interval orwindow and to confirm that the AAA from the prior cycle will havedecayed before administration of another dose of AAA (and chemotherapy).

Dose Limiting Toxicity (DLT) is defined as the occurrence of Grade 4hematologic toxicity, Grade 3 or 4 non-hematologic toxicity includingdiarrhea (despite use of antidiarrheal prophylaxis or glucocorticoids),or nausea and vomiting (despite use of maximal anti-emetics).

TABLE Phase IB dose levels Axitinib Dose (day 1, 1 hr before Levelgemcitabine) Gemcitabine Docetaxel Level −1  5 mg PO 900 mg/m² 75 mg/m²IV days 1, 8 IV day 8 Level 1 10 mg PO 900 mg/m² 75 mg/m² IV days 1, 8IV day 8 Level 2 20 mg PO 900 mg/m² 75 mg/m² IV days 1, 8 IV day 8 Level3 30 mg PO 900 mg/m² 75 mg/m² IV days 1, 8 IV day 8

The materials methods and measurements described herein are not limitingand the same assessments can be made using alternative techniques knownin the art. Similarly, the quantities of AAA and chemotherapeutic agentsused are not limiting and may be subject to adjustment in accordancewith the skill in the art or as dictated by the biomarkers disclosedherein. All cited references are incorporated by reference in theirentirety.

1. A method for enhancing the tumor response to chemotherapy comprising:(a) administering a short-acting anti-angiogenic agent (AAA) to asubject afflicted with a solid tumor, thereby creating a time intervalof increased susceptibility of said tumor to at least onechemotherapeutic agent; (b) administering said at least onechemotherapeutic agent that has the property of activating theASMase/ceramide signaling pathway to said subject at a time point withinsaid interval; thereby enhancing the effect of the at least onechemotherapeutic agent against said tumor compared to thechemotherapeutic agent being used (i) without the anti-angiogenic agentor (ii) at a time point outside the interval.
 2. The method of claim 1further comprising: after the lapse of a time period at least sufficientfor the anti-angiogenic agent to decay, repeating steps (a) and (b). 3.The method of claim 1 wherein presence of the interval is assessed bydetermining a level of ASMase expression and/or activity in said subjectwherein a level of substantial change in expression or activity ofASMase compared to baseline indicates presence of the interval, ordynamic IVIM (intravoxel incoherent motion) DW-MRI wherein a substantialchange in perfusion compared to the baseline perfusion indicatespresence of the interval.
 4. (canceled)
 5. The method of claim 3 whereinactivity of ASMase is assessed by determining levels of a pro-apoptoticceramide.
 6. The method of claim 1 wherein decay of the anti-angiogenicagent is assessed by measuring one or more of serum levels of saidagent, restoration of biologic output of ASMase such that ASMaseactivity will increase upon a subsequent administration of AAA,restoration of biologic output of ceramide, such that ceramide willincrease upon a subsequent administration of AAA, and perfusionalteration upon a subsequent administration of AAA.
 7. The method ofclaim 1 wherein the anti-angiogenic agent has an average plasmahalf-life of up to about 120 hours.
 8. The method of claim 1 wherein theanti-angiogenic agent is at least one selected from the group consistingof cediranib, axitinib, anginex, sunitinib, sorafenib, pazopanib,vatalanib, cabozantinib, ponatinib, lenvatinib, and SU6668.
 9. Themethod of claim 1 wherein the chemotherapeutic agent is selected fromthe group consisting of taxanes, alkylating agents, topoisomeraseinhibitors, endoplasmic reticulum stress inducing agents,antimetabolites, mitotic inhibitors and combinations thereof.
 10. Themethod of claim 1 wherein the chemotherapeutic agent is at least oneselected from the group consisting of chlorambucil, cyclophosphamide,ifosfamide, melphalan, streptozocin, carmustine, lomustine, busulfan,dacarbazine, temozolomide, thiotepa, altretamine, 5-fluorouracil (5-FU),6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine,fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed,daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, ARC, NPC,campothecin, topotecan, 9-nitrocamptothecin, 9-aminocamptothecin,rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, topotecan,amsacrine, etoposide, etoposide phosphate teniposide, doxorubicin,paclitaxel, docetaxel.
 11. (canceled)
 12. The method of claim 11 whereinthe chemotherapeutic agent is selected from the group consisting ofbaccatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol,cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatinIII, 10-deacetyl cephalomannine, and mixtures thereof. 13.-21.(canceled)
 22. The method of claim 1 wherein the tumor is selected fromthe group consisting of adrenal, anal, bile duct, bladder, bone,brain/CNS, breast, cervical, colon/rectum, endometrial, esophageal, eye,gallbladder, gastrointestinal, kidney, heart, head and neck, laryngealand hypopharyngeal, liver, lung, oral mesothelioma, nasopharyngeal,neuroblastoma, ovarian, pancreatic, peritoneal, pituitary, prostate,retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin, smallintestine, stomach, soft tissue sarcoma, rhabdomyosarcoma, testicular,thymus, thyroid, parathyroid, uterine, and vaginal tumors and metastasesthereof. 23.-24. (canceled)
 25. The method of claim 24 wherein the AAAhas a decay period that is about the same as the half-life of the AAA.26.-28. (canceled)
 29. The method of claim 28 wherein thechemotherapeutic agent is administered no more than about 2 hours or nomore than about 1.5 hours or no more than about 1 hour afteradministration of the AAA.
 30. (canceled)
 31. A method for predictingtumor response or monitoring timing and/or efficacy of treatment in apatient afflicted with a malignant tumor to a chemotherapeutic agent,comprising: determining ASMase level or activity in the patientfollowing administration of the chemotherapeutic agent to the patientwherein an increase in said level or activity compared to baseline isindicative of tumor response to the chemotherapeutic agent or confirmsthe efficacy and/or appropriate timing of treatment.
 32. A method forpredicting tumor response or monitoring timing of treatment in a patientto a chemotherapeutic agent, the patient being afflicted with amalignant solid tumor, the method comprising: using dynamic IVIM basedDW-MRI to measure perfusion alterations following administration of thechemotherapeutic agent to determine the extent of rapid perfusiondefects in the tumor vasculature following administration of thechemotherapeutic agent, wherein a statistically significant increase inlevel of said alterations over baseline is indicative of tumor responseto said chemotherapeutic agent or of efficacy or appropriate timing oftreatment.